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MRI Denoising Using Pixel-Wise Threshold Selection
N. Srivastava, G.R. Sahoo, H. U. Voss, S. N. Niogi, J. H. Freed, and M. Srivastava
IEEE Access (In press)
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MRI Denoising Using Pixel-Wise Threshold Selection
N. Srivastava, G.R. Sahoo, H. U. Voss, S. N. Niogi, J. H. Freed, and M. Srivastava
IEEE Access (In press)
Publication #466
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Optimal Wavelet Selection for Signal Denoising
G. R. Sahoo, J. H. Freed, M. Srivastava
IEEE Access 12 45369-45380 (2024)
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Optimal Wavelet Selection for Signal Denoising
G. R. Sahoo, J. H. Freed, M. Srivastava
IEEE Access 12 45369-45380 (2024)
<doi: 10.1109/ACCESS.2024.3377664>
PMID: [In Progress] PMCID:
[In Progress]
Publication #465
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ABSTRACT: Wavelet denoising plays a key role in removing noise from signals and is widely used in many applications. In denoising, selection of the mother wavelet is desirable for maximizing the separation of noise and signal coefficients in the wavelet domain for effective noise thresholding. At present, wavelet selection is carried out in a heuristic manner or using a trial-and-error that is time consuming and prone to error, including human bias. This paper introduces a universal method to select optimal wavelets based on the sparsity of Detail components in the wavelet domain, an empirical approach. A mean of sparsity change ( μsc ) parameter is defined that captures the mean variation of noisy Detail components. The efficacy of the presented method is tested on simulated and experimental signals from Electron Spin Resonance spectroscopy at various SNRs. The results reveal that the μsc values of signal vary abruptly between wavelets, whereas for noise it displays similar values for all wavelets. For low Signal-to-Noise Ratio (SNR) data, the change in μsc between highest and second highest value is ≈8–10% and for high SNR data it is around 5%. The mean of sparsity change increases with the SNR of the signal, which implies that multiple wavelets can be used for denoising a signal, whereas, the signal with low SNR can only be efficiently denoised with a few wavelets. Either a single wavelet or a collection of optimal wavelets (i.e., top five wavelets) should be selected from the highest μsc values. The code is available on GitHub and the signalsciencelab.com website.
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The crystal structure of bacteriophage λ RexA provides novel insights into the DNA binding properties of Rex-like phage exclusion proteins
M. C. Adams, C. J. Schiltz, J. Sun, C. J. Hosford, V. M. Johnson, H. Pan, P. P. Borbat, J. H. Freed, L. C. Thomason, C. Court, D. L. Court, J. S. Chappie
Nucleic Acids Res. 52, 4659-4675 (2024)
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The crystal structure of bacteriophage λ RexA provides novel insights into the DNA binding properties of Rex-like phage exclusion proteins
M. C. Adams, C. J. Schiltz, J. Sun, C. J. Hosford, V. M. Johnson, H. Pan, P. P. Borbat, J. H. Freed, L. C. Thomason, C. Court, D. L. Court, J. S. Chappie
Nucleic Acids Res. 52, 4659-4675 (2024)
Supporting Information
<doi: 10.1093/nar/gkae212>
PMID:
38554102
PMCID:
PMC11077077
Publication #464
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ABSTRACT: RexA and RexB function as an exclusion system that prevents bacteriophage T4rII mutants from growing on Escherichia coli λ phage lysogens. Recent data established that RexA is a non-specific DNA binding protein that can act independently of RexB to bias the λ bistable switch toward the lytic state, preventing conversion back to lysogeny. The molecular interactions underlying these activities are unknown, owing in part to a dearth of structural information. Here, we present the 2.05-Å crystal structure of the λ RexA dimer, which reveals a two-domain architecture with unexpected structural homology to the recombination-associated protein RdgC. Modelling suggests that our structure adopts a closed conformation and would require significant domain rearrangements to facilitate DNA binding. Mutagenesis coupled with electromobility shift assays, limited proteolysis, and double electron–electron spin resonance spectroscopy support a DNA-dependent conformational change. In vivo phenotypes of RexA mutants suggest that DNA binding is not a strict requirement for phage exclusion but may directly contribute to modulation of the bistable switch. We further demonstrate that RexA homologs from other temperate phages also dimerize and bind DNA in vitro. Collectively, these findings advance our mechanistic understanding of Rex functions and provide new evolutionary insights into different aspects of phage biology.
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An analysis of double-quantum coherence ESR in an N-spin system: Analytical expressions and predictions
A. Sinha Roy, J. A. Marohn, J. H. Freed
J. Chem. Phys. 160, 134105 (2024)
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An analysis of double-quantum coherence ESR in an N-spin system: Analytical expressions and predictions
A. Sinha Roy, J. A. Marohn, J. H. Freed
J. Chem. Phys. 160, 134105 (2024)
<doi: 10.1063/5.0200054>
PMID:
38557852
PMCID:
PMC11087869
Publication #463
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ABSTRACT: Electron spin resonance pulsed dipolar spectroscopy (PDS) has become popular in protein 3D structure analysis. PDS studies yield distance distributions between a pair or multiple pairs of spin probes attached to protein molecules, which can be used directly in structural studies or as constraints in theoretical predictions. Double-quantum coherence (DQC) is a highly sensitive and accurate PDS technique to study protein structures in the solid state and under physiologically relevant conditions. In this work, we have derived analytical expressions for the DQC signal for a system with N-dipolar coupled spin-1/2 particles in the solid state. The expressions are integrated over the relevant spatial parameters to obtain closed form DQC signal expressions. These expressions contain the concentration-dependent "instantaneous diffusion" and the background signal. For micromolar and lower concentrations, these effects are negligible. An approximate analysis is provided for cases of finite pulses. The expressions obtained in this work should improve the analysis of DQC experimental data significantly, and the analytical approach could be extended easily to a wide range of magnetic resonance phenomena.
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Differentiating Unimodal and Multimodal Distributions in Pulsed Dipolar Spectroscopy Using Wavelet Transforms
A.Sinha Roy, J. H. Freed, M. Srivastava
Res. Sq. [Preprint] rs.3.rs-3216615 (2023)
Supporting Information
<doi: 10.21203/rs.3.rs-3216615/v1>
PMID:
37577617
PMCID:
PMC10418556
Publication #462
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ABSTRACT: Site directed spin labeling has enabled protein structure determination using electron spin resonance (ESR) pulsed dipolar spectroscopy (PDS). Small details in a distance distribution can be key to understanding important protein structure-function relationships. A major challenge has been to differentiate unimodal and overlapped multimodal distance distributions. They often yield similar distributions and dipolar signals. Current model-free distance reconstruction techniques such as Srivastava-Freed Singular Value Decomposition (SF-SVD) and Tikhonov regularization can suppress these small features in uncertainty and/or error bounds, despite being present. In this work, we demonstrate that continuous wavelet transform (CWT) can distinguish PDS signals from unimodal and multimodal distance distributions. We show that periodicity in CWT representation reflects unimodal distributions, which is masked for multimodal cases. This work is meant as a precursor to a cross-validation technique, which could indicate the modality of the distance distribution.
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Structural insights into perilipin 3 membrane association in response to diacylglycerol accumulation
Y. M. Choi, D. Ajjaji, K. D. Fleming, P. P. Borbat, M. L. Jenkins, B. E. Moeller, S. Fernando, S. R. Bhatia, J. H. Freed, J. E. Burke, A. R. Thiam, M. V. Airola
Nat. Commun. 14 3204 (2023)
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Structural insights into perilipin 3 membrane association in response to diacylglycerol accumulation
Y. M. Choi, D. Ajjaji, K. D. Fleming, P. P. Borbat, M. L. Jenkins, B. E. Moeller, S. Fernando, S. R. Bhatia, J. H. Freed, J. E. Burke, A. R. Thiam, M. V. Airola
Nat. Commun. 14 3204 (2023)
Supporting Information
<doi: 10.1038/s41467-023-38725-w>
PMID:
37268630
PMCID:
PMC10238389
Publication #461
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ABSTRACT: Lipid droplets (LDs) are dynamic organelles that contain an oil core mainly composed of triglycerides (TAG) that is surrounded by a phospholipid monolayer and LD-associated proteins called perilipins (PLINs). During LD biogenesis, perilipin 3 (PLIN3) is recruited to nascent LDs as they emerge from the endoplasmic reticulum. Here, we analyze how lipid composition affects PLIN3 recruitment to membrane bilayers and LDs, and the structural changes that occur upon membrane binding. We find that the TAG precursors phosphatidic acid and diacylglycerol (DAG) recruit PLIN3 to membrane bilayers and define an expanded Perilipin-ADRP-Tip47 (PAT) domain that preferentially binds DAG-enriched membranes. Membrane binding induces a disorder to order transition of alpha helices within the PAT domain and 11-mer repeats, with intramolecular distance measurements consistent with the expanded PAT domain adopting a folded but dynamic structure upon membrane binding. In cells, PLIN3 is recruited to DAG-enriched ER membranes, and this requires both the PAT domain and 11-mer repeats. This provides molecular details of PLIN3 recruitment to nascent LDs and identifies a function of the PAT domain of PLIN3 in DAG binding.
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Thermal degradation of thaumatin at low pH and its prevention using alkyl gallates
B. Pomon, Y. Zhao, A. L. Lai, T. Lin, J. H. Freed, A. Abbaspourrad
Food Hydrocoll. 139, 108544 (2023)
Supporting Information
<doi: 10.1016/j.foodhyd.2023.108544>
PMID:
37546699
PMCID:
PMC10399911
Publication #460
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ABSTRACT: Thaumatin, a potent sweet tasting protein extracted from the Katemfe Plant, is emerging as a natural alternative to synthetic non-nutritive sweeteners and flavor enhancer. As a food additive, its stability within the food matrix during thermal processing is of great interest to the food industry. When heated under neutral or basic conditions, thaumatin was found to lose its sweetness due to protein aggregation caused by sulfhydryl catalyzed disulfide bond interchange. At lower pH, while thaumatin was also found to lose sweetness after heating, it does so at a slower rate and shows more resistance to sweetness loss. SDS-PAGE indicated that thaumatin fragmented into multiple smaller pieces under heating in acidic pH. Using BEMPO-3, a lipophilic spin trap, we were able to detect the presence of a free-radical within the hydrophobic region of the protein during heating. Protein carbonyl content, a byproduct of protein oxidation, also increased upon heating, providing additional evidence for protein cleavage by a radical pathway. Hexyl gallate successfully inhibited the radical generation as well as protein carbonyl formation of thaumatin during heating.
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Membrane Binding Induces Distinct Structural Signatures in the Mouse Complexin–1C–Terminal Domain
E. M. Grasso, M. S. Terakawa, A. L. Lai, Y. X. Xie, T. F. Ramlall, J. H. Freed, and D. Eliezer.
J. Mol. Biol. 435, 167710 (2023)
Supporting Information
<doi: 10.1016/j.jmb.2022.167710>
PMID:
35777466
PMCID:
PMC9794636
Publication #459
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ABSTRACT: Complexins play a critical role in regulating SNARE-mediated exocytosis of synaptic vesicles. Evolutionary divergences in complexin function have complicated our understanding of the role these proteins play in inhibiting the spontaneous fusion of vesicles. Previous structural and functional characterizations of worm and mouse complexins have indicated the membrane curvature-sensing C-terminal domain of these proteins is responsible for differences in inhibitory function. We have characterized the structure and dynamics of the mCpx1 CTD in the absence and presence of membranes and membrane mimetics using NMR, ESR, and optical spectroscopies. In the absence of lipids, the mCpx1 CTD features a short helix near its N-terminus and is otherwise disordered. In the presence of micelles and small unilamellar vesicles, the mCpx1 CTD forms a discontinuous helical structure in its C-terminal 20 amino acids, with no preference for specific lipid compositions. In contrast, the mCpx1 CTD shows distinct compositional preferences in its interactions with large unilamellar vesicles. These studies identify structural divergences in the mCpx1 CTD relative to the wCpx1 CTD in regions that are known to be critical to the wCpx1 CTD's role in inhibiting spontaneous fusion of synaptic vesicles, suggesting a potential structural basis for evolutionary divergences in complexin function.
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Lewis acid-assisted reduction of nitrite to nitric and nitrous oxides via the elusive nitrite radical dianion
V. Hosseininasab, I. M DiMucci, P. Ghosh, J. A. Bertke, S. Chandrasekharan, C. J. Titus, D. Nordlund, J. H. Freed, K. M. Lancaster, T. H. Warren
Nat. Chem. 14, 1265-1269 (2022)
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Lewis acid-assisted reduction of nitrite to nitric and nitrous oxides via the elusive nitrite radical dianion
V. Hosseininasab, I. M DiMucci, P. Ghosh, J. A. Bertke, S. Chandrasekharan, C. J. Titus, D. Nordlund, J. H. Freed, K. M. Lancaster, T. H. Warren
Nat. Chem. 14, 1265-1269 (2022)
Supporting Information
<doi: 10.1038/s41557-022-01025-9>
PMID:
36064970
PMCID:
PMC9633411
Publication #458
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ABSTRACT: Reduction of nitrite anions (NO2–) to nitric oxide (NO), nitrous oxide (N2O) and ultimately dinitrogen (N2) takes place in a variety of environments, including in the soil as part of the biogeochemical nitrogen cycle and in acidified nuclear waste. Nitrite reduction typically takes place within the coordination sphere of a redox-active transition metal. Here we show that Lewis acid coordination can substantially modify the reduction potential of this polyoxoanion to allow for its reduction under non-aqueous conditions (–0.74 V versus NHE). Detailed characterization confirms the formation of the borane-capped radical nitrite dianion (NO22–), which features a N(II) oxidation state. Protonation of the nitrite dianion results in the facile loss of nitric oxide (NO), whereas its reaction with NO results in disproportionation to nitrous oxide (N2O) and nitrite (NO2–). This system connects three redox levels in the global nitrogen cycle and provides fundamental insights into the conversion of NO2– to NO.
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Structural Dynamics by NMR in the Solid State: II. The MOMD Perspective of the Dynamic Structure of Metal-Organic Frameworks Comprising Several Mobile Components
E. Meirovitch, Z. Liang, R. W. Schurko, S. J. Loeb, and J. H. Freed.
J. Phys. Chem. B 126, 2452-2465 (2022)
Supporting Information
<doi: 10.1021/acs.jpcb.1c10120>
PMID:
35333061
PMCID:
PMC9055879
Publication #457
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ABSTRACT: We describe the application of the microscopic-order-macroscopic-disorder (MOMD) approach, developed for the analysis of dynamic 2H NMR lineshapes in the solid state, to unravel interactions among the constituents of metal–organic frameworks (MOFs) that comprise mobile components. MOMD was applied recently to University of Windsor Dynamic Material (UWDM) MOFs with one mobile crown ether per cavity. In this work, we study UWDM-9-d4, which comprises a mobile 2H-labeled phenyl-ring residue along with an isotopically unlabeled 24C8 crown ether. We also study UiO-68-d4, which is structurally similar to UWDM-9-d4 but lacks the crown ether. The physical picture consists of the NMR probe–the C–D bonds of the phenyl-d4 rotor–diffusing locally (diffusion tensor R) in the presence of a local ordering potential, u. For UiO-68-d4, we find it sufficient to expand u in terms of four real Wigner functions, D0|K|L, overall 2–3 kT in magnitude, with R∥ relatively fast, and R⊥; in the (2.8–5.0) × 102 s-1 range. For UWDM-9-d4, u requires only two terms 2–3 kT in magnitude and slower rate constants R∥ and R⊥. In the more crowded macrocycle-containing UWDM-9-d4 cavity, phenyl-d4 dynamics is more isotropic and is described by a simpler ordering potential. This is ascribed to cooperative phenyl-ring/macrocycle motion, which yields a dynamic structure more uniform in character. The experimental 2H spectra used here were analyzed previously with a multi-simple-mode (MSM) approach where several independent simple motional modes are combined. Where possible, similar features have been identified and used to compare the two approaches.
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The N-Terminal Domain of Aβ40-Amyloid Fibril: The MOMD Perspective of its Dynamic Structure from NMR Lineshape Analysis
E. Meirovitch, Z. Liang, and J. H. Freed.
J. Phys. Chem. B 126, 1202-1211 (2022)
Supporting Information
<doi: 10.1021/acs.jpcb.1c10131>
PMID:
35128920
PMCID:
PMC8908910
Publication #456
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ABSTRACT: We have developed the stochastic microscopic‐order‐macroscopic‐disorder (MOMD) approach for elucidating dynamic structures in the solid‐state from 2H NMR lineshapes. In MOMD, the probe experiences an effective/collective motional mode. The latter is described by a potential, u, which represents the local spatial‐restrictions, a local‐motional diffusion tensor, R, and key features of local geometry. Previously we applied MOMD to the well‐structured core domain of the 3‐fold‐symmetric twisted polymorph of the Aβ40‐amyloid fibril. Here, we apply it to the N‐terminal domain of this fibril. We find that the dynamic structures of the two domains are largely similar but differ in the magnitude and complexity of the key physical parameters. This interpretation differs from previous multisimple‐mode (MSM) interpretations of the same experimental data. MSM used for the two domains different combinations of simple motional modes taken to be independent. For the core domain, MOMD and MSM disagree on the character of the dynamic structure. For the N‐terminal domain, they even disagree on whether this chain segment is structurally ordered (MOMD finds that it is), and whether it undergoes a phase transition at 260 K where bulklike water located in the fibril matrix freezes (MOMD finds that it does not). These are major differences associated with an important system. While the MOMD description is a physically sound one, there are drawbacks in the MSM descriptions. The results obtained in this study promote our understanding of the dynamic structure of protein aggregates. Thus, they contribute to the effort to pharmacologically control neurodegenerative disorders believed to be caused by such aggregates.
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Negatively charged residues in the membrane ordering activity of SARS‐CoV‐1 and ‐2 fusion peptides
A.L. Lai and J.H. Freed
Biophys. J. 121, 207-227 (2022)
Supporting Information
<doi: 10.1016/j.bpj.2021.12.024>
PMID:
34929193
PMCID:
PMC8683214
Publication #455
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ABSTRACT: Entry of coronaviruses into host cells is mediated by the viral spike protein. Previously, we identified the bona fide fusion peptides (FPs) for severe acute respiratory syndrome coronavirus ("SARS‐1") and severe acute respiratory syndrome coronavirus‐2 ("SARS‐2") using electron spin resonance spectroscopy. We also found that their FPs induce membrane ordering in a Ca2+‐dependent fashion. Here we study which negatively charged residues in SARS‐1 FP are involved in this binding, to build a topological model and clarify the role of Ca2+. Our systematic mutation study on the SARS‐1 FP shows that all six negatively charged residues contribute to the FP's membrane ordering activity, with D812 the dominant residue. The corresponding SARS‐2 residue D830 plays an equivalent role. We provide a topological model of how the FP binds Ca2+ ions: its two segments FP1 and FP2 each bind one Ca2+. The binding of Ca2+, the folding of FP (both studied by isothermal titration calorimetry experiments), and the ordering activity correlate very well across the mutants, suggesting that the Ca2+ helps the folding of FP in membranes to enhance the ordering activity. Using a novel pseudotyped viral particle‐liposome methodology, we monitored the membrane ordering induced by the FPs in the whole spike protein in its trimer form in real time. We found that the SARS‐1 and SARS‐2 pseudotyped viral particles also induce membrane ordering to the extent that separate FPs do, and mutations of the negatively charged residues also significantly suppress the membrane ordering activity. However, the slower kinetics of the FP ordering activity versus that of the pseudotyped viral particle suggest the need for initial trimerization of the FPs.
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Benchmark Test and Guidelines for DEER/PELDOR Experiments on Nitroxide-Labeled Biomolecules
O. Schiemann, C. A. Heubach, D. Abdullin, K. Ackermann, M. Azarkh, E. G. Bagryanskaya, M. Drescher, B. Endeward, J. H. Freed, L. Galazzo, D. Goldfarb, T. Hett, L. Esteban Hofer, L. Fábregas Ibáñez, E. J. Hustedt, S. Kucher, I. Kuprov, J. E. Lovett, A. Meyer, S. Ruthstein, S. Saxena, S. Stoll, C. R. Timmel, M. Di Valentin, H. S. Mchaourab, T. F. Prisner, B. E. Bode, E. Bordignon, M. Bennati, and G. Jeschke.
J. Am. Chem. Soc. 143, 17875-17890 (2021)
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Benchmark Test and Guidelines for DEER/PELDOR Experiments on Nitroxide-Labeled Biomolecules
O. Schiemann, C. A. Heubach, D. Abdullin, K. Ackermann, M. Azarkh, E. G. Bagryanskaya, M. Drescher, B. Endeward, J. H. Freed, L. Galazzo, D. Goldfarb, T. Hett, L. Esteban Hofer, L. Fábregas Ibáñez, E. J. Hustedt, S. Kucher, I. Kuprov, J. E. Lovett, A. Meyer, S. Ruthstein, S. Saxena, S. Stoll, C. R. Timmel, M. Di Valentin, H. S. Mchaourab, T. F. Prisner, B. E. Bode, E. Bordignon, M. Bennati, and G. Jeschke.
J. Am. Chem. Soc. 143, 17875-17890 (2021)
Supporting Information
<doi: 10.1021/jacs.1c07371>
PMID:
34664948
PMCID:
PMC11253894
Publication #454
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ABSTRACT: Distance distribution information obtained by pulsed dipolar EPR spectroscopy provides an important contribution to many studies in structural biology. Increasingly, such information is used in integrative structural modeling, where it delivers unique restraints on the width of conformational ensembles. In order to ensure reliability of the structural models and of biological conclusions, we herein define quality standards for sample preparation and characterization, for measurements of distributed dipole–dipole couplings between paramagnetic labels, for conversion of the primary time‐domain data into distance distributions, for interpreting these distributions, and for reporting results. These guidelines are substantiated by a multi‐laboratory benchmark study and by analysis of data sets with known distance distribution ground truth. The study and the guidelines focus on proteins labeled with nitroxides and on double electron–electron resonance (DEER aka PELDOR) measurements and provide suggestions on how to proceed analogously in other cases.
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Highly Basic Clusters in the Herpes Simplex Virus 1 Nuclear Egress Complex Drive Membrane Budding by Inducing Lipid Ordering
M. K. Thorsen, A. Lai, D. P. Hoogerheide, G. C. L. Wong, J. H. Freed, and E. E. Heldwein.
mBio 12 e0154821 (2021)
Supporting Information
<doi: 10.1128/mBio.01548-21>
PMID:
34425706
PMCID:
PMC8406295
Publication #453
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ABSTRACT: During replication of herpesviruses, capsids escape from the nucleus into the cytoplasm by budding at the inner nuclear membrane. This unusual process is mediated by the viral nuclear egress complex (NEC) that deforms the membrane around the capsid by oligomerizing into a hexagonal, membrane‐bound scaffold. Here, we found that highly basic membrane‐proximal regions (MPRs) of the NEC alter lipid order by inserting into the lipid headgroups and promote negative Gaussian curvature. We also find that the electrostatic interactions between the MPRs and the membranes are essential for membrane deformation. One of the MPRs is phosphorylated by a viral kinase during infection, and the corresponding phosphomimicking mutations block capsid nuclear egress. We show that the same phosphomimicking mutations disrupt the NEC‐membrane interactions and inhibit NEC‐mediated budding in vitro, providing a biophysical explanation for the in vivo phenomenon. Our data suggest that the NEC generates negative membrane curvature by both lipid ordering and protein scaffolding and that phosphorylation acts as an off switch that inhibits the membrane‐budding activity of the NEC to prevent capsid‐less budding.
IMPORTANCE Herpesviruses are large viruses that infect nearly all vertebrates and some invertebrates and cause lifelong infections in most of the world's population. During replication, herpesviruses export their capsids from the nucleus into the cytoplasm by an unusual mechanism in which the viral nuclear egress complex (NEC) deforms the nuclear membrane around the capsid. However, how membrane deformation is achieved is unclear. Here, we show that the NEC from herpes simplex virus 1, a prototypical herpesvirus, uses clusters of positive charges to bind membranes and order membrane lipids. Reducing the positive charge or introducing negative charges weakens the membrane deforming ability of the NEC. We propose that the virus employs electrostatics to deform nuclear membrane around the capsid and can control this process by changing the NEC charge through phosphorylation. Blocking NEC-membrane interactions could be exploited as a therapeutic strategy.
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Theory and Least Squares Fitting of CW ESR Saturation Spectra Using the MOMD Model
P. Gupta, B. Dzikovski, and J. H. Freed
Appl. Magn. Reson. 53 699-715 (2021)
<doi: 10.1007/s00723-021-01390-7>
PMID:
35431460
PMCID:
PMC9012167
Publication #452
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ABSTRACT: CW saturation experiments are widely used in ESR studies of relaxation processes in proteins and lipids. We develop the theory of saturation in ESR spectra in terms of its close relation with that of 2D‐ELDOR. Our treatment of saturation is then based on the microscopic order macroscopic disorder (MOMD) model and can be used to fit the full CW saturation spectrum, rather than fitting just the peak–peak amplitude as a function of microwave field B1 as is commonly done. This requires fewer experiments to yield effects on T1, as well as provides a more extensive dynamic structural picture, for example, for scanning experiments on different protein sites. The code is released as a publicly available software package in Python that can be used to fit CW saturation spectra from biological samples of interest.
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Dph3 Enables Aerobic Diphthamide Biosynthesis by Donating One Iron Atom to Transform a [3Fe-4S] to a [4Fe-4S] Cluster in Dph1-Dph2
Y. Zhang, D. Su, B. Dzikovski, S. H. Majer, R. Coleman, S. Chandrasekaran, M. K. Fenwick, B. R. Crane, K. M. Lancaster, J. H. Freed, and H. Lin.
J. Am. Chem. Soc. 143, 9314-9319 (2021)
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Dph3 Enables Aerobic Diphthamide Biosynthesis by Donating One Iron Atom to Transform a [3Fe-4S] to a [4Fe-4S] Cluster in Dph1-Dph2
Y. Zhang, D. Su, B. Dzikovski, S. H. Majer, R. Coleman, S. Chandrasekaran, M. K. Fenwick, B. R. Crane, K. M. Lancaster, J. H. Freed, and H. Lin.
J. Am. Chem. Soc. 143, 9314-9319 (2021)
Supporting Information
<doi: 10.1021/jacs.1c03956>
PMID:
34154323
PMCID:
PMC8251694
Publication #451
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ABSTRACT: All radical S‐adenosylmethionine (radical‐SAM) enzymes, including the noncanonical radical‐SAM enzyme diphthamide biosynthetic enzyme Dph1–Dph2, require at least one [4Fe–4S](Cys)3 cluster for activity. It is well‐known in the radical‐SAM enzyme community that the [4Fe–4S](Cys)3 cluster is extremely air‐sensitive and requires strict anaerobic conditions to reconstitute activity in vitro. Thus, how such enzymes function in vivo in the presence of oxygen in aerobic organisms is an interesting question. Working on yeast Dph1–Dph2, we found that consistent with the known oxygen sensitivity, the [4Fe–4S] cluster is easily degraded into a [3Fe–4S] cluster. Remarkably, the small iron‐containing protein Dph3 donates one Fe atom to convert the [3Fe–4S] cluster in Dph1–Dph2 to a functional [4Fe–4S] cluster during the radical‐SAM enzyme catalytic cycle. This mechanism to maintain radical‐SAM enzyme activity in aerobic environments is likely general, and Dph3‐like proteins may exist to keep other radical‐SAM enzymes functional in aerobic environments.
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Extraction of Weak Spectroscopic Signals with High Fidelity: Examples from ESR
M. Srivastava, B. Dzikovski, and J. H. Freed.
J. Phys. Chem. A 125, 4480-4487 (2021)
Supporting Information
<doi: 10.1021/acs.jpca.1c02241>
PMID:
34009996
PMCID:
PMC8317606
Publication #450
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ABSTRACT: Noise impedes experimental studies by reducing signal resolution and/or suppressing weak signals. Signal averaging and filtering are the primary methods used to reduce noise, but they have limited effectiveness and lack capabilities to recover signals at low signal‐to‐noise ratios (SNRs). We utilize a wavelet transform‐based approach to effectively remove noise from spectroscopic data. The wavelet denoising method we use is a significant improvement on standard wavelet denoising approaches. We demonstrate its power in extracting signals from noisy spectra on a variety of signal types ranging from hyperfine lines to overlapped peaks to weak peaks overlaid on strong ones, drawn from electron‐spin‐resonance spectroscopy. The results show that one can accurately extract details of complex spectra, including retrieval of very weak ones. It accurately recovers signals at an SNR of ˜1 and improves the SNR by about 3 orders of magnitude with high fidelity. Our examples show that one is now able to address weaker SNR signals much better than by previous methods. This new wavelet approach can be successfully applied to other spectroscopic signals.
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SARS-CoV-2 Fusion Peptide has a Greater Membrane Perturbating Effect than SARS-CoV with Highly Specific Dependence on Ca2+
A. L. Lai and J. H. Freed.
J. Mol. Biol. 433, 166946 (2021)
<doi: 10.1016/j.jmb.2021.166946>
PMID:
33744314
PMCID:
PMC7969826
Publication #449
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ABSTRACT: Coronaviruses are a major infectious disease threat, and include the zoonotic‐origin human pathogens SARS‐CoV‐2, SARS‐CoV, and MERS‐CoV (SARS‐2, SARS‐1, and MERS). Entry of coronaviruses into host cells is mediated by the spike (S) protein. In our previous ESR studies, the local membrane ordering effect of the fusion peptide (FP) of various viral glycoproteins including the S of SARS‐1 and MERS has been consistently observed. We previously determined that the sequence immediately downstream from the S2′ cleavage site is the bona fide SARS‐1 FP. In this study, we used sequence alignment to identify the SARS‐2 FP, and studied its membrane ordering effect. Although there are only three residue differences, SARS‐2 FP induces even greater membrane ordering than SARS‐1 FP, possibly due to its greater hydrophobicity. This may be a reason that SARS‐2 is better able to infect host cells. In addition, the membrane binding enthalpy for SARS‐2 is greater. Both the membrane ordering of SARS‐2 and SARS‐1 FPs are dependent on Ca2+, but that of SARS‐2 shows a greater response to the presence of Ca2+. Both FPs bind two Ca2+ ions as does SARS‐1 FP, but the two Ca2+ binding sites of SARS‐2 exhibit greater cooperativity. This Ca2+ dependence by the SARS‐2 FP is very ion‐specific. These results show that Ca2+ is an important regulator that interacts with the SARS‐2 FP and thus plays a significant role in SARS‐2 viral entry. This could lead to therapeutic solutions that either target the FP‐calcium interaction or block the Ca2+ channel.
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Microsecond dynamics in proteins by two-dimensional ESR. II. Addressing computational challenges
P. Gupta, K. Chaudhari, and J. H. Freed.
J. Chem. Phys. 154, 084115 (2021)
Supporting Information
<doi: 10.1063/5.0042441>
PMID:
33639766
PMCID:
PMC7928224
Publication #448
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ABSTRACT: Two‐dimensional electron‐electron double resonance (2D‐ELDOR) provides extensive insight into molecular motions. Recent developments permitting experiments at higher frequencies (95 GHz) provide molecular orientational resolution, enabling a clearer description of the nature of the motions. In previous work, we provided simulations for the case of domain motions within proteins that are themselves slowly tumbling in a solution. In order to perform these simulations, it was found that the standard approach of solving the relevant stochastic Liouville equation using the efficient Lanczos algorithm for this case breaks down, so algorithms were employed that rely on the Arnoldi iteration. While they lead to accurate simulations, they are very time‐consuming. In this work, we focus on a variant known as the rational Arnoldi algorithm. We show that this can achieve a significant reduction in computation time. The stochastic Liouville matrix, which is of very large dimension, N, is first reduced to a much smaller dimension, m, e.g., from N ˜ O(104) to m ˜ 60, that spans the relevant Krylov subspace from which the spectrum is predicted. This requires the selection of the m frequency shifts to be utilized. A method of adaptive shift choice is introduced to optimize this selection. We also find that these procedures help in optimizing the pruning procedure that greatly reduces the dimension of the initial N dimensional stochastic Liouville matrix in such subsequent computations.
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Microsecond Exchange Processes Studied by Two‐Dimensional ESR at 95 GHz
B. Dzikovski, V. V. Khramtsov, S. Chandrasekaran, C. Dunnam, M. Shah, and J. H. Freed.
J. Am. Chem. Soc. 142, 21368-21381 (2020)
Supporting Information
<doi: 10.1021/jacs.0c09469>
PMID:
33305945
PMCID:
PMC7810061
Publication #447
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ABSTRACT: Exchange processes which include conformational change, protonation/deprotonation, and binding equilibria are routinely studied by 2D exchange NMR techniques, where information about the exchange of nuclei between environments with different NMR shifts is obtained from the development of cross‐peaks. Whereas 2D NMR enables the real time study of millisecond and slower exchange processes, 2D ESR in the form of 2D‐ELDOR (two‐dimensional electron‐electron double resonance) has the potential for such studies over the nanosecond to microsecond real time scales. Cross‐peak development due to chemical exchange has been seen previously for semiquinones in ESR, but this is not possible for most common ESR probes, such as nitroxides, studied at typical ESR frequencies because, unlike NMR, the exchanging states yield ESR signals that are not resolved from each other within their respective line widths. But at 95 GHz, it becomes possible to resolve them in many cases because of the increased g‐factor resolution. The 95 GHz instrumental developments occurring at ACERT now enable such studies. We demonstrate these new capabilities in two studies: (A) the protonation/deprotonation process for a pH‐sensitive imidazoline spin label in aqueous solution where the exchange rate and the population ratio of the exchanging states are controlled by the concentration and pH of the buffer solution, respectively, and (B) a nitroxide radical partitioning between polar (aqueous) and nonpolar (phospholipid) environments in multilamellar lipid vesicles, where the cross‐peak development arises from the exchange of the nitroxide between the two phases. This work represents the first example of the observation and analysis of cross‐peaks arising from chemical exchange processes involving nitroxide spin labels.
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Engineered chemotaxis core signaling units indicate a constrained kinase-off state
A. R. Muok, T. K. Chua, M. Srivastava, W. Yang, Z. Maschmann, P. P. Borbat, J. Chong, S. Zhang, J. H. Freed, A. Briegel, B. R. Crane.
Sci. Signal. 13, eabc1328 (2020)
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Engineered chemotaxis core signaling units indicate a constrained kinase-off state
A. R. Muok, T. K. Chua, M. Srivastava, W. Yang, Z. Maschmann, P. P. Borbat, J. Chong, S. Zhang, J. H. Freed, A. Briegel, B. R. Crane.
Sci. Signal. 13, eabc1328 (2020)
Supporting Information
<doi: 10.1126/scisignal.abc1328>
PMID:
33172954
PMCID:
PMC7790435
Publication #446
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ABSTRACT: Bacterial chemoreceptors, the histidine kinase CheA, and the coupling protein CheW form transmembrane molecular arrays with remarkable sensing properties. The receptors inhibit or stimulate CheA kinase activity depending on the presence of attractants or repellants, respectively. We engineered chemoreceptor cytoplasmic regions to assume a trimer of receptor dimers configuration that formed well‐defined complexes with CheA and CheW and promoted a CheA kinase‐off state. These mimics of core signaling units were assembled to homogeneity and investigated by site‐directed spin‐labeling with pulse‐dipolar electron‐spin resonance spectroscopy (PDS), small‐angle x‐ray scattering, targeted protein cross‐linking, and cryo–electron microscopy. The kinase‐off state was especially stable, had relatively low domain mobility, and associated the histidine substrate and docking domains with the kinase core, thus preventing catalytic activity. Together, these data provide an experimentally restrained model for the inhibited state of the core signaling unit and suggest that chemoreceptors indirectly sequester the kinase and substrate domains to limit histidine autophosphorylation.
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Structural Dynamics by NMR in the Solid State: The Unified MOMD Perspective Applied to Organic Frameworks with Interlocked Molecules
E. Meirovitch, Z. Liang, and J. H. Freed.
J. Phys. Chem. B 124, 6225-6235 (2020)
Supporting Information
<doi: 10.1021/acs.jpcb.0c03687>
PMID:
32584038
PMCID:
PMC7666760
Publication #445
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ABSTRACT: The microscopic-order-macroscopic-disorder (MOMD) approach for NMR lineshape analysis has been applied to the University of Windsor Dynamic Materials (UWDM) of types 1, 2, α-3, β-3, and 5, which are metal–organic frameworks (MOFs) comprising mobile mechanically interlocked molecules (MIMs). The mobile MIM components are selectively deuterated crown ether macrocycles – 24C6, 22C6, and B24C6. Their motion is described in MOMD by an effective/collective dynamic mode characterized by a diffusion tensor, R, a restricting/ordering potential, u, expanded in the Wigner rotation matrix elements, D0,KL, and features of local geometry. Experimental 2H lineshapes are available over 220 K (on average) and in some cases 320 K. They are reproduced with axial R, u given by the terms D0,02 and D0,|2|2, and established local geometry. For UWDM of types 1, β-3, and 5, where the macrocycle resides in a relatively loose space, u is in the 1–3 kT, R∥ in the (1.0–2.5) × 106 s–1, and R⊥ in the (0.4–2.5) × 104 s–1 range; the deuterium atom is bonded to a carbon atom with tetrahedral coordination character. For UWDM of types 2 and α-3, where the macrocycle resides in a much tighter space, a substantial change in the symmetry of u and the coordination character of the 2H-bonded carbon are detected at higher temperatures. The activation energies for R∥ and R⊥ are characteristic of each system. The MOMD model is general; effective/collective dynamic modes are treated. The characteristics of motion, ordering, and geometry are physically well-defined; they differ from case to case in extent and symmetry but not in essence. Physical clarity and consistency provide new insights. A previous interpretation of the same experimental data used models consisting of collections of independent simple motions. These models are specific to each case and temperature. Within their scope, generating consistent physical pictures and comparing cases are difficult; possible collective modes are neglected.
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Conformational Dynamics in Extended RGD-Containing Peptides
W. R. Lindemann, A. J. Mijalis, J. L. Alonso, P. P. Borbat, J. H. Freed, M. A. Arnaout, B. L. Pentelute, and J. H. Ortony.
Biomacromolecules 21, 2786-2794 (2020)
Supporting Information
<doi: 10.1021/acs.biomac.0c00506>
PMID:
32469507
PMCID:
PMC7388056
Publication #444
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ABSTRACT: RGD is a prolific example of a tripeptide used in biomaterials for cell adhesion, but the potency of free or surface-bound RGD tripeptide is orders-of-magnitude less than the RGD domain within natural proteins. We designed a set of peptides with varying lengths, composed of fragments of fibronectin protein whose central three residues are RGD, in order to vary their conformational behavior without changing the binding site's chemical environment. With these peptides, we measure the conformational dynamics and transient structure of the active site. Our studies reveal how flanking residues affect conformational behavior and integrin binding. We find that disorder of the binding site is important to the potency of RGD peptides and that transient hydrogen bonding near the RGD site affects both the energy landscape roughness of the peptides and peptide binding. This phenomenon is independent of longer-range folding interactions and helps explain why short binding sequences, including RGD itself, do not fully replicate the integrin-targeting properties of extracellular matrix proteins. Our studies reinforce that peptide binding is a holistic event and fragments larger than those directly involved in binding should be considered in the design of peptide epitopes for functional biomaterials.
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Microsecond dynamics in proteins by two-dimensional ESR: Predictions
P. Gupta, Z. Liang, and J. H. Freed.
J. Chem. Phys. 152, 214112 (2020)
Supporting Information
<doi: 10.1063/5.0008094>
PMID:
32505151
PMCID:
PMC7863697
Publication #443
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ABSTRACT: Two-dimensional electron-electron double resonance (2D-ELDOR) provides extensive insight into molecular motions. Recent developments permitting experiments at higher frequencies (95 GHz) provide molecular orientational resolution, enabling a clearer description of the nature of the motions. In this work, simulations are provided for the example of domain motions within proteins that are themselves slowly tumbling in solution. These show the nature of the exchange cross-peaks that are predicted to develop in real time from such domain motions. However, we find that the existing theoretical methods for computing 2D-ELDOR experiments over a wide motional range begin to fail seriously when applied to very slow motions characteristic of proteins in solution. One reason is the failure to obtain accurate eigenvectors and eigenvalues of the complex symmetric stochastic Liouville matrices describing the experiment when computed by the efficient Lanczos algorithm in the range of very slow motion. Another, perhaps more serious, issue is that these matrices are "non-normal," such that for the very slow motional range even rigorous diagonalization algorithms do not yield the correct eigenvalues and eigenvectors. We have employed algorithms that overcome both these issues and lead to valid 2D-ELDOR predictions even for motions approaching the rigid limit. They are utilized to describe the development of cross-peaks in 2D-ELDOR at 95 GHz for a particular case of domain motion.
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George K. Fraenkel, Electron Spin Resonance Pioneer
J.H. Freed.
In Pioneers of Magnetic Resonance. Strom, E. T., Mainz, V. V., Eds. American Chemical Society: Washington, DC, ACS Symposium Series, 2020; Volume 1349, Chapter 8, pp. 137-154.
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George K. Fraenkel, Electron Spin Resonance Pioneer
J.H. Freed.
In Pioneers of Magnetic Resonance. Strom, E. T., Mainz, V. V., Eds. American Chemical Society: Washington, DC, ACS Symposium Series, 2020; Volume 1349, Chapter 8, pp. 137-154.
<doi: 10.1021/bk-2020-1349.ch008>
PMID: [None-book] PMCID:
[None-book]
Publication #442
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ABSTRACT: George K. Fraenkel (1921-2009), although less known today, was one of the leading pioneers in the development and use of Electron Spin Resonance (ESR) techniques for studying the structure and dynamical interactions of molecules. Together with his students he developed high-sensitivity, high-resolution spectrometers that enabled them to pioneer the study of ESR in free radicals in solution. His work provided breakthroughs that led to advances in several fields of chemistry, and laid the foundations for later research on the properties of biological systems. This chapter provides a thematic overview, arranged chronologically, of his various studies. Short descriptions are provided of his achievements in these areas, based on his most important papers, so that present-day researchers can appreciate the extent of his accomplishments.
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High-yield production in E. coli and characterization of full-length functional p13II protein from human T-cell leukemia virus type 1
E. R. Georgieva, P. P. Borbat, C. Fanouraki, J. H. Freed.
Protein Expr. Purif. 173, 105659 (2020)
Supporting Information
<doi: 10.1016/j.pep.2020.105659>
PMID:
32360379
PMCID:
PMC7266171
Publication #441
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ABSTRACT: Human T-cell leukemia virus type 1 is an oncovirus that causes aggressive adult T-cell leukemia but is also responsible for severe neurodegenerative and endocrine disorders. Combatting HTLV-1 infections requires a detailed understanding of the viral mechanisms in the host. Therefore, in vitro studies of important virus-encoded proteins would be critical. Our focus herein is on the HTLV-1-encoded regulatory protein p13II, which interacts with the inner mitochondrial membrane, increasing its permeability to cations (predominantly potassium, K+). Thereby, this protein affects mitochondrial homeostasis. We report on our progress in developing specific protocols for heterologous expression of p13II in E. coli, and methods for its purification and characterization. We succeeded in producing large quantities of highly-pure full-length p13II, deemed to be its fully functional form. Importantly, our particular approach based on the fusion of ubiquitin to the p13II C-terminus was instrumental in increasing the persistently low expression of soluble p13II in its native form. We subsequently developed approaches for protein spin labeling and a conformation study using double electron-electron resonance (DEER) spectroscopy and a fluorescence-based cation uptake assay for p13II in liposomes. Our DEER results point to large protein conformation changes occurring upon transition from the soluble to the membrane-bound state. The functional assay on p13II-assisted transport of thallium (Tl+) through the membrane, wherein Tl+ substituted for K+, suggests transmembrane potential involvement in p13II function. Our study lays the foundation for expansion of in vitro functional and structural investigations on p13II and would aid in the development of structure-based protein inhibitors and markers.
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Ca2+ Ions Promote Fusion of Middle East Respiratory Syndrome Coronavirus with Host Cells and Increase Infectivity
M. R. Straus, T. Tang, A. L. Lai, A. Flegel, M. Bidon, J. H. Freed, S. Daniel, G. R. Whittaker.
J. Virol. 94, e00426-20 (2020)
Supporting Information
<doi: 10.1128/JVI.00426-20>
PMID:
32295925
PMCID:
PMC7307142
Publication #440
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ABSTRACT: Fusion with, and subsequent entry into, the host cell is one of the critical steps in the life cycle of enveloped viruses. For Middle East respiratory syndrome coronavirus (MERS-CoV), the spike (S) protein is the main determinant of viral entry. Proteolytic cleavage of the S protein exposes its fusion peptide (FP), which initiates the process of membrane fusion. Previous studies on the related severe acute respiratory syndrome coronavirus (SARS-CoV) FP have shown that calcium ions (Ca2+) play an important role in fusogenic activity via a Ca2+ binding pocket with conserved glutamic acid (E) and aspartic acid (D) residues. SARS-CoV and MERS-CoV FPs share a high sequence homology, and here, we investigated whether Ca2+ is required for MERS-CoV fusion by screening a mutant array in which E and D residues in the MERS-CoV FP were substituted with neutrally charged alanines (A). Upon verifying mutant cell surface expression and proteolytic cleavage, we tested their ability to mediate pseudoparticle (PP) infection of host cells in modulating Ca2+ environments. Our results demonstrate that intracellular Ca2+ enhances MERS-CoV wild-type (WT) PP infection by approximately 2-fold and that E891 is a crucial residue for Ca2+ interaction. Subsequent electron spin resonance (ESR) experiments revealed that this enhancement could be attributed to Ca2+ increasing MERS-CoV FP fusion-relevant membrane ordering. Intriguingly, isothermal calorimetry showed an approximate 1:1 MERS-CoV FP to Ca2+ ratio, as opposed to an 1:2 SARS-CoV FP to Ca2+ ratio, suggesting significant differences in FP Ca2+ interactions of MERS-CoV and SARS-CoV FP despite their high sequence similarity.
IMPORTANCE Middle East respiratory syndrome coronavirus (MERS-CoV) is a major emerging infectious disease with zoonotic potential and has reservoirs in dromedary camels and bats. Since its first outbreak in 2012, the virus has repeatedly transmitted from camels to humans, with 2,468 confirmed cases causing 851 deaths. To date, there are no efficacious drugs and vaccines against MERS-CoV, increasing its potential to cause a public health emergency. In order to develop novel drugs and vaccines, it is important to understand the molecular mechanisms that enable the virus to infect host cells. Our data have found that calcium is an important regulator of viral fusion by interacting with negatively charged residues in the MERS-CoV FP region. This information can guide therapeutic solutions to block this calcium interaction and also repurpose already approved drugs for this use for a fast response to MERS-CoV outbreaks.
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Calcium Ions Directly Interact with the Ebola Virus Fusion Peptide To Promote Structure–Function Changes That Enhance Infection
L. Nathan, A. L. Lai, J. K. Millet, M. R. Straus, J. H. Freed, G. R. Whittaker, and S. Daniel.
ACS Infect. Dis. 6, 250-260 (2020)
Supporting Information
<doi: 10.1021/acsinfecdis.9b00296>
PMID:
31746195
PMCID:
PMC7040957
Publication #439
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ABSTRACT: Ebola virus disease is a serious global health concern given its periodic occurrence, high lethality, and the lack of approved therapeutics. Certain drugs that alter intracellular calcium, particularly in endolysosomes, have been shown to inhibit Ebola virus infection; however, the underlying mechanism is unknown. Here, we provide evidence that Zaire ebolavirus (EBOV) infection is promoted in the presence of calcium as a result of the direct interaction of calcium with the EBOV fusion peptide (FP). We identify the glycoprotein residues D522 and E540 in the FP as functionally critical to EBOV's interaction with calcium. We show using spectroscopic and biophysical assays that interactions of the fusion peptide with Ca2+directly targets the Ebola virus fusion peptide and influences its conformation. As these residues are highly conserved across the Filoviridae, calcium's impact on fusion, and subsequently infectivity, is a key interaction that can be leveraged for developing strategies to defend against Ebola infection. This mechanistic insight provides a rationale for the use of calcium-interfering drugs already approved by the FDA as therapeutics against Ebola and enables further development of novel drugs to combat the virus.
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The asymmetric function of Dph1–Dph2 heterodimer in diphthamide biosynthesis
M. Dong, E. E. Dando, I. Kotliar, X. Su, B. Dzikovski, J. H. Freed, and H. Lin.
J. Biol. Inorg. Chem. 24, 777-782 (2019)
<doi: 10.1007/s00775-019-01702-0>
PMID:
31463593
PMCID:
PMC6893874
Publication #438
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ABSTRACT: Diphthamide, the target of diphtheria toxin, is a post-translationally modified histidine residue found in archaeal and eukaryotic translation elongation factor 2 (EF2). In the first step of diphthamide biosynthesis, a [4Fe–4S] cluster-containing radical SAM enzyme, Dph1–Dph2 heterodimer in eukaryotes or Dph2 homodimer in archaea, cleaves S-adenosylmethionine and transfers the 3-amino-3-carboxypropyl group to EF2. It was demonstrated previously that for the archaeal Dph2 homodimer, only one [4Fe–4S] cluster is necessary for the in vitro activity. Here, we demonstrate that for the eukaryotic Dph1–Dph2 heterodimer, the [4Fe–4S] cluster-binding cysteine residues in each subunit are required for diphthamide biosynthesis to occur in vivo. Furthermore, our in vitro reconstitution experiments with Dph1–Dph2 mutants suggested that the Dph1 cluster serves a catalytic role, while the Dph2 cluster facilitates the reduction of the Dph1 cluster by the physiological reducing system Dph3/Cbr1/NADH. Our results reveal the asymmetric functional roles of the Dph1–Dph2 heterodimer and may help to understand how the Fe–S clusters in radical SAM enzymes are reduced in biology.
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Local ordering and dynamics in anisotropic media by magnetic resonance: from liquid crystals to proteins
E. Meirovitch & J. H. Freed.
Liq. Cryst. 47, 1926-1954 (2020)
<doi: 10.1080/02678292.2019.1622158>
PMID:
32435078
PMCID:
PMC7239324
Publication #437
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ABSTRACT: Magnetic resonance methods have been used extensively for over 50 years to elucidate molecular structure and dynamics of liquid crystals (LCs), providing information quite unique in its rigour and extent. The ESR- or NMR-active probe is often a solute molecule reporting on characteristics associated with the surrounding (LC) medium, which exerts the spatial restrictions on the probe. The theoretical approaches developed for LCs are applicable to anisotropic media in general. Of particular interest is the interior space of a globular protein labelled, e.g. with a nitroxide moiety or a 15N–1H bond. The ESR or NMR label plays the role of the probe and the internal protein surroundings the role of the anisotropic medium. A general feature of the restricted motions is the local ordering, i.e. the nature, magnitude and symmetry of the spatial restraints exerted at the site of the moving probe. This property is the main theme of the present review article. We outline its treatment in our work from both the theoretical and the experimental points of view, highlighting the new physical insights gained. Our illustrations include studies on thermotropic (nematic and smectic) and lyotropic liquid crystals formed by phospholipids, in addition to studies of proteins.
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Insights into histidine kinase activation mechanisms from the monomeric blue light sensor EL346
I. Dikiy, U. R. Edupuganti, R. R. Abzalimov, P. P. Borbat, M. Srivastava, J. H. Freed, and K. H. Gardner.
Proc. Natl. Acad. Sci. 116, 4963-4972 (2019)
Supporting Information
<doi: 10.1073/pnas.1813586116>
PMID:
30808807
PMCID:
PMC6421462
Publication #436
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SIGNIFICANCE: All living things must sense and react to their environment. Many single-celled organisms do so by using two-component systems, most simply consisting of a sensor histidine kinase and a response regulator. These systems are involved in pathogenicity pathways and can be targeted by new antibiotics. However, the molecular mechanisms used by histidine kinases to translate sensing into responses are not well understood. To probe this general question, we apply a combination of biophysical techniques to a monomeric histidine kinase that senses blue light to determine the structural changes occurring upon activation. We find these changes to be similar to those predicted for the common dimeric histidine kinases, illustrating that the mechanism of activation is conserved regardless of oligomeric state.
ABSTRACT: Translation of environmental cues into cellular behavior is a necessary process in all forms of life. In bacteria, this process frequently involves two-component systems in which a sensor histidine kinase (HK) autophosphorylates in response to a stimulus before subsequently transferring the phosphoryl group to a response regulator that controls downstream effectors. Many details of the molecular mechanisms of HK activation are still unclear due to complications associated with the multiple signaling states of these large, multidomain proteins. To address these challenges, we combined complementary solution biophysical approaches to examine the conformational changes upon activation of a minimal, blue-light–sensing histidine kinase from Erythrobacter litoralis HTCC2594, EL346. Our data show that multiple conformations coexist in the dark state of EL346 in solution, which may explain the enzyme's residual dark-state activity. We also observe that activation involves destabilization of the helices in the dimerization and histidine phosphotransfer-like domain, where the phosphoacceptor histidine resides, and their interactions with the catalytic domain. Similar light-induced changes occur to some extent even in constitutively active or inactive mutants, showing that light sensing can be decoupled from activation of kinase activity. These structural changes mirror those inferred by comparing X-ray crystal structures of inactive and active HK fragments, suggesting that they are at the core of conformational changes leading to HK activation. More broadly, our findings uncover surprising complexity in this simple system and allow us to outline a mechanism of the multiple steps of HK activation.
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Singular Value Decomposition Method To Determine Distance Distributions in Pulsed Dipolar Electron Spin Resonance: II. Estimating Uncertainty
M. Srivastava and J. H. Freed.
J. Phys. Chem. A 123, 359-370 (2019)
Supporting Information
<doi: 10.1021/acs.jpca.8b07673>
PMID:
30525624
PMCID:
PMC6372347
Publication #434
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ABSTRACT: This paper is a continuation of the method introduced by Srivastava and Freed (2017) that is a new method based on truncated singular value decomposition (TSVD) for obtaining physical results from experimental signals without any need for Tikhonov regularization or other similar methods that require a regularization parameter. We show here how to estimate the uncertainty in the SVD-generated solutions. The uncertainty in the solution may be obtained by finding the minimum and maximum values over which the solution remains converged. These are obtained from the optimum range of singular value contributions, where the width of this region depends on the solution point location (e.g., distance) and the signal-to-noise ratio (SNR) of the signal. The uncertainty levels typically found are very small with substantial SNR of the (denoised) signal, emphasizing the reliability of the method. With poorer SNR, the method is still satisfactory but with greater uncertainty, as expected. Pulsed dipolar electron spin resonance spectroscopy experiments are used as an example, but this TSVD approach is general and thus applicable to any similar experimental method wherein singular matrix inversion is needed to obtain the physically relevant result. We show that the Srivastava–Freed TSVD method along with the estimate of uncertainty can be effectively applied to pulsed dipolar electron spin resonance signals with SNR > 30, and even for a weak signal (e.g., SNR ≈ 3) reliable results are obtained by this method, provided the signal is first denoised using wavelet transforms (WavPDS).
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Cofactors are essential constituents of stable and seeding-active tau fibrils
Y. Fichou, Y. Lin, J. N. Rauch, M. Vigers, Z. Zeng, M. Srivastava, T. J. Keller, J. H. Freed, K. S. Kosik, and S. Han.
Proc. Natl. Acad. Sci. 115, 13234-13239 (2018)
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Cofactors are essential constituents of stable and seeding-active tau fibrils
Y. Fichou, Y. Lin, J. N. Rauch, M. Vigers, Z. Zeng, M. Srivastava, T. J. Keller, J. H. Freed, K. S. Kosik, and S. Han.
Proc. Natl. Acad. Sci. 115, 13234-13239 (2018)
Supporting Information
<doi: 10.1073/pnas.1810058115>
PMID:
30538196
PMCID:
PMC6310788
Publication #433
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SIGNIFICANCE: The tau protein is involved in Alzheimer's and other neurodegenerative diseases, where the location, morphology, and quantity of amyloid fibrils composed of tau correlate with the disease type and stage. While tau fibrillary aggregates have been colocalized in brains together with several cofactors, their role in fibril formation, structure, and seeding has been largely neglected. We show that seeding of tau aggregation is facilitated by polyanionic cofactors, and that seeded or recombinant mature fibrils depolymerize into monomers when their cofactor is removed. We show that cofactor-assisted seeding with mouse brain-derived tau fibrils yielded tau fibrils with distinct and narrowed structural properties compared with heparin-induced fibrils, suggesting that the fibrillar templates tuned the structure of the seeded fibril.
ABSTRACT: Amyloid fibrils are cross-β–rich aggregates that are exceptionally stable forms of protein assembly. Accumulation of tau amyloid fibrils is involved in many neurodegenerative diseases, including Alzheimer's disease (AD). Heparin-induced aggregates have been widely used and assumed to be a good tau amyloid fibril model for most biophysical studies. Here we show that mature fibrils made of 4R tau variants, prepared with heparin or RNA, spontaneously depolymerize and release monomers when their cofactors are removed. We demonstrate that the cross-β-sheet assembly formed in vitro with polyanion addition is unstable at room temperature. We furthermore demonstrate high seeding capacity with transgenic AD mouse brain-extracted tau fibrils in vitro that, however, is exhausted after one generation, while supplementation with RNA cofactors resulted in sustained seeding over multiple generations. We suggest that tau fibrils formed in brains are supported by unknown cofactors and inhere higher-quality packing, as reflected in a more distinct conformational arrangement in the mouse fibril-seeded, compared with heparin-induced, tau fibrils. Our study suggests that the role of cofactors in tauopathies is a worthy focus of future studies, as they may be viable targets for diagnosis and therapeutics.
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Phenyl-Ring Dynamics in Amyloid Fibrils and Proteins: The Microscopic-Order-Macroscopic-Disorder Perspective
E. Meirovitch, Z. Liang, and J. H. Freed.
J. Phys. Chem. B 122, 8675-8684 (2018)
Supporting Information
<doi: 10.1021/acs.jpcb.8b06330>
PMID:
30141954
PMCID:
PMC6174686
Publication #432
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ABSTRACT: We have developed the microscopic-order-macroscopic-disorder (MOMD) approach for studying internal mobility in polycrystalline proteins with 2H lineshape analysis. The motion itself is expressed by a diffusion tensor, R, the local spatial restraints by a potential, u, and the "local geometry" by the relative orientation of the model-related and nuclear magnetic resonance-related tensors. Here, we apply MOMD to phenyl-ring dynamics in several Αβ40-amyloid-fibrils, and the villin headpiece subdomain (HP36). Because the available data are limited in extent and sensitivity, we adjust u and R in the relevant parameter ranges, fixing the "local geometry" in accordance with standard stereochemistry. This yields a physically well-defined and consistent picture of phenyl-ring dynamics, enabling comparison between different systems. In the temperature range of 278–308 K, u has a strength of (1.7–1.8) kT and a rhombicity of (2.4–2.6) kT, and R has components of 5.0 × 102 ≤ R⊥ ≤ 2.0 × 103 s–1 and 6.3 × 105 ≤ R∥ ≤ 2.0 × 106 s–1. At 278 K, fibril hydration increases the axiality of both u and R; HP36 hydration has a similar effect at 295 K, reducing R⊥ considerably. The D23N mutation slows down the motion of the probe; Αβ40 polymorphism affects both this motion and the related local potential. The present study identifies the impact of various factors on phenyl-ring mobility in amyloid fibrils and globular proteins; the difference between the two protein forms is considerable. The distinctive impact of hydration on phenyl-ring motion and previously studied methyl-group motion is also examined. The 2H lineshapes considered here were analyzed previously with various multi-simple-mode (MSM) models, where several simple motional modes are combined. The MOMD and MSM interpretations differ in essence.
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A facile approach for the in vitro assembly of multimeric membrane transport proteins
E. A. Riederer, P. J. Focke, E. R. Georgieva, N. Akyuz, K. Matulef, P. P. Borbat, J. H. Freed, S. C. Blanchard, O. Boudker, and F. I. Valiyaveetil.
eLife 7, e36478 (2018)
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A facile approach for the in vitro assembly of multimeric membrane transport proteins
E. A. Riederer, P. J. Focke, E. R. Georgieva, N. Akyuz, K. Matulef, P. P. Borbat, J. H. Freed, S. C. Blanchard, O. Boudker, and F. I. Valiyaveetil.
eLife 7, e36478 (2018)
Supporting Information
<doi: 10.7554/eLife.36478>
PMID:
29889023
PMCID:
PMC6025958
Publication #431
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ABSTRACT: Membrane proteins such as ion channels and transporters are frequently homomeric. The homomeric nature raises important questions regarding coupling between subunits and complicates the application of techniques such as FRET or DEER spectroscopy. These challenges can be overcome if the subunits of a homomeric protein can be independently modified for functional or spectroscopic studies. Here, we describe a general approach for in vitro assembly that can be used for the generation of heteromeric variants of homomeric membrane proteins. We establish the approach using GltPh, a glutamate transporter homolog that is trimeric in the native state. We use heteromeric GltPh transporters to directly demonstrate the lack of coupling in substrate binding and demonstrate how heteromeric transporters considerably simplify the application of DEER spectroscopy. Further, we demonstrate the general applicability of this approach by carrying out the in vitro assembly of VcINDY, a Na+-coupled succinate transporter and CLC-ec1, a Cl-/H+ antiporter.
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Open and Closed Form of Maltose Binding Protein in Its Native and Molten Globule State As Studied by Electron Paramagnetic Resonance Spectroscopy
B. Selmke, P. P. Borbat, C. Nickolaus, R. Varadarajan, J. H. Freed, and W. E. Trommer.
Biochemistry 57, 5507-5512 (2018)
Supporting Information
<doi: 10.1021/acs.biochem.8b00322>
PMID:
30004675
PMCID:
PMC6211580
Publication #430
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ABSTRACT: An intensively investigated intermediate state of protein folding is the molten globule (MG) state, which contains secondary but hardly any tertiary structure. In previous work, we have determined the distances between interacting spins within maltose binding protein (MBP) in its native state using continuous wave and double electron–electron resonance (DEER) electron paramagnetic resonance (EPR) spectroscopy. Seven double mutants had been employed to investigate the structure within the two domains of MBP. DEER data nicely corroborated the previously available X-ray data. Even in its MG state, MBP is known to still bind its ligand maltose. We therefore hypothesized that there must be a defined structure around the binding pocket of MBP already in the absence of tertiary structure. Here we have investigated the functional and structural difference between native and MG state in the open and closed form with a new set of MBP mutants. In these, the spin-label positions were placed near the active site. Binding of its ligands leads to a conformational change from open to closed state, where the two domains are more closely together. The complete set of MBP mutants was analyzed at pH 3.2 (MG) and pH 7.4 (native state) using double-quantum coherence EPR. The values were compared with theoretical predictions of distances between the labels in biradicals constructed by molecular modeling from the crystal structures of MBP in open and closed form and were found to be in excellent agreement. Measurements show a defined structure around the binding pocket of MBP in MG, which explains maltose binding. A new and important finding is that in both states ligand-free MBP can be found in open and closed form, while ligand-bound MBP appears only in closed form because of maltose binding.
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Site-Specific Incorporation of a Cu2+ Spin Label into Proteins for Measuring Distances by Pulsed Dipolar Electron Spin Resonance Spectroscopy
G. E. Merz, P. P. Borbat, A. R. Muok, M. Srivastava, D. N. Bunck, J. H. Freed, and B. R. Crane.
J. Phys. Chem. B 122, 9443-9451 (2018)
Supporting Information
<doi: 10.1021/acs.jpcb.8b05619>
PMID:
30222354
PMCID:
PMC6215709
Publication #429
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ABSTRACT: Pulsed dipolar electron spin resonance spectroscopy (PDS) is a powerful tool for measuring distances in solution-state macromolecules. Paramagnetic metal ions, such as Cu2+, are used as spin probes because they can report on metalloprotein features and can be spectroscopically distinguished from traditional nitroxide (NO)-based labels. Here, we demonstrate site-specific incorporation of Cu2+ into non-metalloproteins through the use of a genetically encodable non-natural amino acid, 3-pyrazolyltyrosine (PyTyr). We first incorporate PyTyr in cyan fluorescent protein to measure Cu2+-to-NO distances and examine the effects of solvent conditions on Cu2+ binding and protein aggregation. We then apply the method to characterize the complex formed by the histidine kinase CheA and its target response regulator CheY. The X-ray structure of CheY–PyTyr confirms Cu labeling at PyTyr but also reveals a secondary Cu site. Cu2+-to-NO and Cu2+-to-Cu2+ PDS measurements of CheY–PyTyr with nitroxide-labeled CheA provide new insights into the conformational landscape of the phosphotransfer complex and have implications for kinase regulation.
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MOMD Analysis of NMR Line Shapes from Aβ-Amyloid Fibrils: A New Tool for Characterizing Molecular Environments in Protein Aggregates
E. Meirovitch, Z. Liang, and J. H. Freed.
J. Phys. Chem. B 122, 4793-4801 (2018)
Supporting Information
<doi: 10.1021/acs.jpcb.8b02181>
PMID:
29624402
PMCID:
PMC5945340
Publication #428
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ABSTRACT: The microscopic-order-macroscopic-disorder (MOMD) approach for 2H NMR line shape analysis is applied to dry and hydrated 3-fold- and 2-fold-symmetric amyloid-Aβ40 fibrils and protofibrils of the D23N mutant. The methyl moieties of L17, L34, V36 (C–CD3), and M35 (S–CD3) serve as probes. Experimental 2H spectra acquired previously in the 147–310 K range are used. MOMD describes local probe motion as axial diffusion (R tensor) in the presence of a potential, u, which represents the spatial restrictions exerted by the molecular surroundings. We find that R∥ = (0.2–3.3) × 104 s–1, R⊥ = (2.2–2.5) × 102 s–1, and R is tilted from the 2H quadrupolar tensor at 60–75°. The strength of u is in the (2.0–2.4) kT range; its rhombicity is substantial. The only methyl moieties affected by fibril hydration are those of M35, located at fibril interfaces. The associated local potentials change form abruptly around 260 K, where massive water freezing occurs. An independent study revealed unfrozen "tightly-peptide-bound" water residing at the interfaces of the 3-fold-symmetric Aβ40 fibrils and at the interfaces of the E22G and E22Δ Aβ40-mutant fibrils. Considering this to be the case in general for Aβ40-related fibrils, the following emerges. The impact of water freezing is transmitted selectively to the fibril structure through interactions with tightly-peptide-bound water, in this case of M35 methyl moieties. The proof that such waters reside at the interfaces of the 2-fold-symmetric fibril, and the protofibril of the D23N mutant, is new. MOMD provides information on the surroundings of the NMR probe directly via the potential, u, which is inherent to the model; a prior interpretation of the same experimental data does so partially and indirectly (see below). Thus, MOMD analysis of NMR line shapes as applied to amyloid fibrils/protein aggregates emerges as a consistent new tool for elucidating the properties of, and processes associated with, molecular environments in the fibril.
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Structural basis for membrane anchoring and fusion regulation of the herpes simplex virus fusogen gB
R. S. Cooper, E. R. Georgieva, P. P. Borbat, J. H. Freed, and E. E. Heldwein.
Nat. Struct. Mol. Biol. 25, 416-424 (2018)
Supporting Information
<doi: 10.1038/s41594-018-0060-6>
PMID:
29728654
PMCID:
PMC5942590
Publication #427
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ABSTRACT: Viral fusogens merge viral and cell membranes during cell penetration. Their ectodomains drive fusion by undergoing large-scale refolding, but little is known about the functionally important regions located within or near the membrane. Here we report the crystal structure of full-length glycoprotein B (gB), the fusogen from herpes simplex virus, complemented by electron spin resonance measurements. The membrane-proximal (MPR), transmembrane (TMD), and cytoplasmic (CTD) domains form a uniquely folded trimeric pedestal beneath the ectodomain, which balances dynamic flexibility with extensive, stabilizing membrane interactions. The postfusion conformation of the ectodomain suggests that the CTD likewise adopted the postfusion form. However, hyperfusogenic mutations, which destabilize the prefusion state of gB, target key interfaces and structural motifs that reinforce the observed CTD structure. Thus, a similar CTD structure must stabilize gB in its prefusion state. Our data suggest a model for how this dynamic, membrane-dependent 'clamp' controls the fusogenic refolding of gB.
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Protein dynamics in the solid-state from 2H NMR lineshape analysis. III. MOMD in the presence of Magic Angle Spinning
E. Meirovitch, Z. Liang, J. H. Freed.
Solid State Nucl. Magn. Reson. 89, 35-44 (2018)
<doi: 10.1016/j.ssnmr.2017.11.001>
PMID:
29208317
PMCID:
PMC5772661
Publication #426
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ABSTRACT: We report on a new approach to the analysis of dynamic NMR lineshapes from polycrystalline (i.e., macroscopically disordered) samples in the presence of Magic Angle Spinning (MAS). This is an application of the Stochastic Liouville Equation developed by Freed and co-workers for treating restricted (i.e., microscopically ordered) motions. The 2H nucleus in an internally-mobile C–CD3 moiety serves as a prototype probe. The acronym is 2H/MOMD/MAS, where MOMD stands for "microscopic-order-macroscopic-disorder." The key elements describing internal motions – their type, the local spatial restrictions, and related features of local geometry – are treated in MOMD generally, within their rigorous three-dimensional tensorial requirements. Based on this representation a single physically well-defined model of local motion has the capability of reproducing experimental spectra. There exist other methods for analyzing dynamic 2H/MAS spectra which advocate simple motional modes. Yet, to reproduce satisfactorily the experimental lineshapes, one has either to use unusual parameter values, or combine several simple motional modes. The multi-simple-mode reasoning assumes independence of the constituent modes, features ambiguity as different simple modes may be used, renders inter-system comparison difficult as the overall models differ, and makes possible model-improvement only by adding yet another simple mode, i.e., changing the overall model. 2H/MOMD/MAS is free of such limitations and inherently provides a clear physical interpretation. These features are illustrated. The advantage of 2H/MOMD/MAS in dealing with sensitive but hardly investigated slow-motional lineshapes is demonstrated by applying it to actual experimental data. The results differ from those obtained previously with a two-site exchange scheme that yielded unusual parameters.
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Synthesis and Solution-Phase Characterization of Sulfonated Oligothioetheramides
J. S. Brown, Y. M. Acevedo, G. D. He, J. H. Freed, P. Clancy, and C. A. Alabi.
Macromolecules 50, 8731-8738 (2017)
Supporting Information
<doi: 10.1021/acs.macromol.7b01915>
PMID:
29386690
PMCID:
PMC5788177
Publication #425
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ABSTRACT: Nature has long demonstrated the importance of chemical sequence to induce structure and tune physical interactions. Investigating macromolecular structure and dynamics is paramount to understand macromolecular binding and target recognition. To that end, we have synthesized and characterized flexible sulfonated oligothioetheramides (oligoTEAs) by variable temperature pulse field gradient (PFG) NMR, double electron–electron resonance (DEER), and molecular dynamics (MD) simulations to capture their room temperature structure and dynamics in water. We have examined the contributions of synthetic length (2–12mer), pendant group charge, and backbone hydrophobicity. We observe significant entropic collapse, driven in part by backbone hydrophobicity. Analysis of individual monomer contributions revealed larger changes due to the backbone compared to pendant groups. We also observe screening of intramolecular electrostatic repulsions. Finally, we comment on the combination of DEER and PFG NMR measurements via Stokes–Einstein–Sutherland diffusion theory. Overall, this sensitive characterization holds promise to enable de novo development of macromolecular structure and sequence–structure–function relationships with flexible, but biologically functional macromolecules.
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Organometallic and radical intermediates reveal mechanism of diphthamide biosynthesis
M. Dong, V. Kathiresan, M. K. Fenwick, A. T. Torelli, Y. Zhang, J. D. Caranto, B. Dzikovski, A. Sharma, K. M. Lancaster, J. H. Freed, S. E. Ealick, B. M. Hoffman, and H. Lin.
Science 359, 1247-1250 (2018)
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Organometallic and radical intermediates reveal mechanism of diphthamide biosynthesis
M. Dong, V. Kathiresan, M. K. Fenwick, A. T. Torelli, Y. Zhang, J. D. Caranto, B. Dzikovski, A. Sharma, K. M. Lancaster, J. H. Freed, S. E. Ealick, B. M. Hoffman, and H. Lin.
Science 359, 1247-1250 (2018)
Supporting Information
<doi: 10.1126/science.aao6595>
PMID:
29590073
PMCID:
PMC6066404
Publication #424
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ABSTRACT: Diphthamide biosynthesis involves a carbon-carbon bond-forming reaction catalyzed by a radical S-adenosylmethionine (SAM) enzyme that cleaves a carbon-sulfur (C–S) bond in SAM to generate a 3-amino-3-carboxypropyl (ACP) radical. Using rapid freezing, we have captured an organometallic intermediate with an iron-carbon (Fe–C) bond between ACP and the enzyme's [4Fe-4S] cluster. In the presence of the substrate protein, elongation factor 2, this intermediate converts to an organic radical, formed by addition of the ACP radical to a histidine side chain. Crystal structures of archaeal diphthamide biosynthetic radical SAM enzymes reveal that the carbon of the SAM C–S bond being cleaved is positioned near the unique cluster Fe, able to react with the cluster. Our results explain how selective C–S bond cleavage is achieved in this radical SAM enzyme.
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Dipolar Spectroscopy – Single-Resonance Methods
P. P. Borbat and J. H. Freed.
eMagRes 6 425-462 (2017)
<doi: 10.1002/9780470034590.emrstm1519>
PMID: [None - Book] PMCID:
[None - Book]
Publication #423
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ABSTRACT: A range of powerful techniques based on pulse EPR spectroscopies is used extensively for measuring electron spin dipolar couplings between native or introduced paramagnetic reporters, from which accurate distances, and in certain cases molecular orientations, can be determined to aid in solving biomolecular structures. Notably, several versatile pulse EPR methods of high sensitivity were developed over the past two decades. The two main groups of pulse methods are represented by single- and double-resonance techniques, which together constitute pulse dipolar EPR spectroscopy (PDS). We describe the first group by outlining the basic principles and illustrate key techniques with several examples from the literature, as well as some newly produced for purposes of this chapter. Specifically, we describe double-quantum coherence (DQC) methods for robust selection of the dipolar coupling by filtering out undesired spin coherence pathways via phase cycling, and we also discuss single-resonance methods such as four- and five-pulse single-quantum coherence (SQC) methods, all of which benefit from maximizing the spectral coverage. The common features and conceptual differences between single- and double-resonance methods are discussed. In addition, the extensions of modern DQC and SQC methods to two-dimensional correlation spectroscopy for eliciting orientational information are included. Other types of 'single-frequency' PDS methods, not all of which are based on principles behind fully coherent single-resonance methods, are also discussed to show the diversity of PDS methods.
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Singular Value Decomposition Method to Determine Distance Distributions in Pulsed Dipolar Electron Spin Resonance
M. Srivastava and J. H. Freed.
J. Phys. Chem. Lett. 8, 5648-5655 (2017)
Supporting Information
<doi: 10.1021/acs.jpclett.7b02379>
PMID:
29099190
PMCID:
PMC5708871
Publication #422
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ABSTRACT: Regularization is often utilized to elicit the desired physical results from experimental data. The recent development of a denoising procedure yielding about 2 orders of magnitude in improvement in SNR obviates the need for regularization, which achieves a compromise between canceling effects of noise and obtaining an estimate of the desired physical results. We show how singular value decomposition (SVD) can be employed directly on the denoised data, using pulse dipolar electron spin resonance experiments as an example. Such experiments are useful in measuring distances and their distributions, P(r) between spin labels on proteins. In noise-free model cases exact results are obtained, but even a small amount of noise (e.g., SNR = 850 after denoising) corrupts the solution. We develop criteria that precisely determine an optimum approximate solution, which can readily be automated. This method is applicable to any signal that is currently processed with regularization of its SVD analysis.
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The SARS-CoV Fusion Peptide Forms an Extended Bipartite Fusion Platform that Perturbs Membrane Order in a Calcium-Dependent Manner
A. L. Lai, J. K. Millet, S. Daniel, J. H. Freed, and G. R. Whittaker.
J. Mol. Biol. 429, 3875-3892 (2017)
<doi: 10.1016/j.jmb.2017.10.017>
PMID:
29056462
PMCID:
PMC5705393
Publication #421
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ABSTRACT: Coronaviruses (CoVs) are a major infectious disease threat and include the pathogenic human pathogens of zoonotic origin: severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV). Entry of CoVs into host cells is mediated by the viral spike (S) protein, which is structurally categorized as a class I viral fusion protein, within the same group as influenza virus and HIV. However, S proteins have two distinct cleavage sites that can be activated by a much wider range of proteases. The exact location of the CoV fusion peptide (FP) has been disputed. However, most evidence suggests that the domain immediately downstream of the S2′ cleavage site is the FP (amino acids 798–818 SFIEDLLFNKVTLADAGFMKQY for SARS-CoV, FP1). In our previous electron spin resonance spectroscopic studies, the membrane-ordering effect of influenza virus, HIV, and Dengue virus FPs has been consistently observed. In this study, we used this effect as a criterion to identify and characterize the bona fide SARS-CoV FP. Our results indicate that both FP1 and the region immediately downstream (amino acids 816–835 KQYGECLGDINARDLICAQKF, FP2) induce significant membrane ordering. Furthermore, their effects are calcium dependent, which is consistent with in vivo data showing that calcium is required for SARS-CoV S-mediated fusion. Isothermal titration calorimetry showed a direct interaction between calcium cations and both FPs. This Ca2+-dependency membrane ordering was not observed with influenza FP, indicating that the CoV FP exhibits a mechanistically different behavior. Membrane-ordering effects are greater and penetrate deeper into membranes when FP1 and FP2 act in a concerted manner, suggesting that they form an extended fusion "platform."
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Mechanistic Insight into the Photocontrolled Cationic Polymerization of Vinyl Ethers
Q. Michaudel, T. Chauviré, V. Kottisch, M. J. Supej, K. J. Stawiasz, L. Shen, W. R. Zipfel, H. D. Abruña, J. H. Freed, and B. P. Fors.
J. Am. Chem. Soc. 139, 15530-15538 (2017)
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Mechanistic Insight into the Photocontrolled Cationic Polymerization of Vinyl Ethers
Q. Michaudel, T. Chauviré, V. Kottisch, M. J. Supej, K. J. Stawiasz, L. Shen, W. R. Zipfel, H. D. Abruña, J. H. Freed, and B. P. Fors.
J. Am. Chem. Soc. 139, 15530-15538 (2017)
Supporting Information
<doi: 10.1021/jacs.7b09539>
PMID:
28985061
PMCID:
PMC5806523
Publication #420
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ABSTRACT: The mechanism of the recently reported photocontrolled cationic polymerization of vinyl ethers was investigated using a variety of catalysts and chain-transfer agents (CTAs) as well as diverse spectroscopic and electrochemical analytical techniques. Our study revealed a complex activation step characterized by one-electron oxidation of the CTA. This oxidation is followed by mesolytic cleavage of the resulting radical cation species, which leads to the generation of a reactive cation–this species initiates the polymerization of the vinyl ether monomer–and a dithiocarbamate radical that is likely in equilibrium with the corresponding thiuram disulfide dimer. Reversible addition–fragmentation type degenerative chain transfer contributes to the narrow dispersities and control over chain growth observed under these conditions. Finally, the deactivation step is contingent upon the oxidation of the reduced photocatalyst by the dithiocarbamate radical concomitant with the production of a dithiocarbamate anion that caps the polymer chain end. The fine-tuning of the electronic properties and redox potentials of the photocatalyst in both the excited and the ground states is necessary to obtain a photocontrolled system rather than simply a photoinitiated system. The elucidation of the elementary steps of this process will aid the design of new catalytic systems and their real-world applications.
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Unique Structural Features of Membrane-Bound C-Terminal Domain Motifs Modulate Complexin Inhibitory Function
D. Snead, A. L. Lai, R. T. Wragg, D. A. Parisotto, T. F. Ramlall, J. S. Dittman, J. H. Freed, and D. Eliezer.
Front. Mol. Neurosci. 10, 154 (2017)
Supporting Information
<doi: 10.3389/fnmol.2017.00154>
PMID:
28596722
PMCID:
PMC5442187
Publication #419
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ABSTRACT: Complexin is a small soluble presynaptic protein that interacts with neuronal SNARE proteins in order to regulate synaptic vesicle exocytosis. While the SNARE-binding central helix of complexin is required for both the inhibition of spontaneous fusion and the facilitation of synchronous fusion, the disordered C-terminal domain (CTD) of complexin is specifically required for its inhibitory function. The CTD of worm complexin binds to membranes via two distinct motifs, one of which undergoes a membrane curvature dependent structural transition that is required for efficient inhibition of neurotransmitter release, but the conformations of the membrane-bound motifs remain poorly characterized. Visualizing these conformations is required to clarify the mechanisms by which complexin membrane interactions regulate its function. Here, we employ optical and magnetic resonance spectroscopy to precisely define the boundaries of the two CTD membrane-binding motifs and to characterize their conformations. We show that the curvature dependent amphipathic helical motif features an irregular element of helical structure, likely a pi-bulge, and that this feature is important for complexin inhibitory function in vivo.
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Substrate-Dependent Cleavage Site Selection by Unconventional Radical S-Adenosylmethionine Enzymes in Diphthamide Biosynthesis
M. Dong, M. Horitani, B. Dzikovski, J. H. Freed, S. E. Ealick, B. M. Hoffman, and H. Lin.
J. Am. Chem. Soc. 139, 5680-5683 (2017)
Supporting Information
<doi: 10.1021/jacs.7b01712>
PMID:
28383907
PMCID:
PMC5664936
Publication #418
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ABSTRACT: S-Adenosylmethionine (SAM) has a sulfonium ion with three distinct C-S bonds. Conventional radical SAM enzymes use a [4Fe-4S] cluster to cleave homolytically the C5′,adenosine-S bond of SAM to generate a 5′-deoxyadenosyl radical, which catalyzes various downstream chemical reactions. Radical SAM enzymes involved in diphthamide biosynthesis, such as Pyrococcus horikoshii Dph2 (PhDph2) and yeast Dph1-Dph2 instead cleave the Cγ,Met-S bond of methionine to generate a 3-amino-3-carboxylpropyl radical. We here show radical SAM enzymes can be tuned to cleave the third C-S bond to the sulfonium sulfur by changing the structure of SAM. With a decarboxyl SAM analogue (dc-SAM), PhDph2 cleaves the Cmethyl-S bond, forming 5′-deoxy-5′-(3-aminopropylthio) adenosine (dAPTA, 1). The methyl cleavage activity, like the cleavage of the other two C-S bonds, is dependent on the presence of a [4Fe-4S]+ cluster. Electron-nuclear double resonance and mass spectroscopy data suggests that mechanistically one of the S atoms in the [4Fe-4S] cluster captures the methyl group from dc-SAM, forming a distinct EPR-active intermediate, which can transfer the methyl group to nucleophiles such as dithiothreitol. This reveals the [4Fe-4S] cluster in a radical SAM enzyme can be tuned to cleave any one of the three bonds to the sulfonium sulfur of SAM or analogues, and is the first demonstration a radical SAM enzyme could switch from an Fe-based one electron transfer reaction to a S-based two electron transfer reaction in a substrate-dependent manner. This study provides an illustration of the versatile reactivity of Fe-S clusters.
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Key features of an Hsp70 chaperone allosteric landscape revealed by ion-mobility native mass spectrometry and double electron-electron resonance
A. L. Lai, E. M. Clerico, M. E. Blackburn, N. A. Patel, C. V. Robinson, P. P. Borbat, J. H. Freed, and L. M. Gierasch.
J. Biol. Chem. 292, 8773-8785 (2017)
Supporting Information
<doi: 10.1074/jbc.M116.770404>
PMID:
28428246
PMCID:
PMC5448104
Publication #417
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ABSTRACT: Proteins are dynamic entities that populate conformational ensembles, and most functions of proteins depend on their dynamic character. Allostery, in particular, relies on ligand-modulated shifts in these conformational ensembles. Hsp70s are allosteric molecular chaperones with conformational landscapes that involve large rearrangements of their two domains (viz. the nucleotide-binding domain and substrate-binding domain) in response to adenine nucleotides and substrates. However, it remains unclear how the Hsp70 conformational ensemble is populated at each point of the allosteric cycle and how ligands control these populations. We have mapped the conformational species present under different ligand-binding conditions throughout the allosteric cycle of the Escherichia coli Hsp70 DnaK by two complementary methods, ion-mobility mass spectrometry and double electron-electron resonance. Our results obtained under biologically relevant ligand-bound conditions confirm the current picture derived from NMR and crystallographic data of domain docking upon ATP binding and undocking in response to ADP and substrate. Additionally, we find that the helical lid of DnaK is a highly dynamic unit of the structure in all ligand-bound states. Importantly, we demonstrate that DnaK populates a partially docked state in the presence of ATP and substrate and that this state represents an energy minimum on the DnaK allosteric landscape. Because Hsp70s are emerging as potential drug targets for many diseases, fully mapping an allosteric landscape of a molecular chaperone like DnaK will facilitate the development of small molecules that modulate Hsp70 function via allosteric mechanisms.
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A New Wavelet Denoising Method for Experimental Time-Domain Signals: Pulsed Dipolar Electron Spin Resonance
M. Srivastava, E. R. Georgieva, and J. H. Freed.
J. Phys. Chem. A 121 2452-2465 (2017)
Supporting Information
<doi: 10.1021/acs.jpca.7b0018>
PMID:
28257206
PMCID:
PMC5553325
Publication #416
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ABSTRACT: We adapt a new wavelet-transform-based method of denoising experimental signals to pulse-dipolar electron-spin resonance spectroscopy (PDS). We show that signal averaging times of the time-domain signals can be reduced by as much as 2 orders of magnitude, while retaining the fidelity of the underlying signals, in comparison with noiseless reference signals. We have achieved excellent signal recovery when the initial noisy signal has an SNR ≳ 3. This approach is robust and is expected to be applicable to other time-domain spectroscopies. In PDS, these time-domain signals representing the dipolar interaction between two electron spin labels are converted into their distance distribution functions P(r), usually by regularization methods such as Tikhonov regularization. The significant improvements achieved by using denoised signals for this regularization are described. We show that they yield P(r)'s with more accurate detail and yield clearer separations of respective distances, which is especially important when the P(r)'s are complex. Also, longer distance P(r)'s, requiring longer dipolar evolution times, become accessible after denoising. In comparison to standard wavelet denoising approaches, it is clearly shown that the new method (WavPDS) is superior.
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Stability and Conformation of a Chemoreceptor HAMP Domain Chimera Correlates with Signaling Properties
N. Sukomon, J. Widom, P. P. Borbat, J. H. Freed, and B. R. Crane.
Biophys. J. 112, 1383-1395 (2017)
Supporting Information
<doi: 10.1016/j.bpj.2017.02.037>
PMID:
28402881
PMCID:
PMC5390053
Publication #415
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ABSTRACT: HAMP domains are dimeric, four-helix bundles that transduce conformational signals in bacterial receptors. Genetic studies of the Escherichia coli serine receptor (Tsr) provide an opportunity to understand HAMP conformational behavior in terms of functional output. To increase its stability, the Tsr HAMP domain was spliced into a poly-HAMP unit from the Pseudomonas aeruginosa Aer2 receptor. Within the chimera, the Tsr HAMP undergoes a thermal melting transition at a temperature much lower than that of the Aer2 HAMP domains. Pulse-dipolar electron spin resonance spectroscopy and site-specific spin-labeling confirm that the Tsr HAMP maintains a four-helix bundle. Pulse-dipolar electron spin resonance spectroscopy was also used to study three well-characterized HAMP mutational phenotypes: those that cause flagella rotation that is counterclockwise (CCW) A and kinase-off; CCW B and also kinase-off; and, clockwise (CW) and kinase-on. Conformational properties of the three HAMP variants support a biphasic model of dynamic bundle stability, but also indicate distinct conformational changes within the helix bundle. Functional kinase-on (CW) and kinase-off (CCW A) states differ by concerted changes in the positions of spin-label sites at the base of the bundle. Opposite shifts in the subunit separation distances of neighboring residues at the C-termini of the α1 and α2 helices are consistent with a helix scissors motion or a gearbox rotational model of HAMP activation. In the drastic kinase-off lesion of CCW B, the α1 helices unfold and the α2 helices form a tight two-helix coiled-coil. The substitution of a critical residue in the Tsr N-terminal linker or control cable reduces conformational heterogeneity at the N-terminus of α1 but does not affect structure at the C-terminus of α2. Overall, the data suggest that transitions from on- to off-states involve decreased motional amplitudes of the Tsr HAMP coupled with helix rotations and movements toward a two-helix packing mode.
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Structure-Function Studies Link Class II Viral Fusogens with the Ancestral Gamete Fusion Protein HAP2
J. F. Pinello, A. L. Lai, J. K. Millet, D. Cassidy-Hanley, J. H. Freed, and T. G. Clark.
Curr. Biol. 27, 651-660 (2017)
Supporting Information
<doi: 10.1016/j.cub.2017.01.049>
PMID:
28238660
PMCID:
PMC5393271
Publication #414
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ABSTRACT: The conserved transmembrane protein, HAP2/GCS1, has been linked to fertility in a wide range of taxa and is hypothesized to be an ancient gamete fusogen. Using template-based structural homology modeling, we now show that the ectodomain of HAP2 orthologs from Tetrahymena thermophila and other species adopt a protein fold remarkably similar to the dengue virus E glycoprotein and related class II viral fusogens. To test the functional significance of this predicted structure, we developed a flow-cytometry-based assay that measures cytosolic exchange across the conjugation junction to rapidly probe the effects of HAP2 mutations in the Tetrahymena system. Using this assay, alterations to a region in and around a predicted "fusion loop" in T. thermophila HAP2 were found to abrogate membrane pore formation in mating cells. Consistent with this, a synthetic peptide corresponding to the HAP2 fusion loop was found to interact directly with model membranes in a variety of biophysical assays. These results raise interesting questions regarding the evolutionary relationships of class II membrane fusogens and harken back to a long-held argument that eukaryotic sex arose as the byproduct of selection for the horizontal transfer of a "selfish" genetic element from cell to cell via membrane fusion.
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Signature of an aggregation-prone conformation of tau
N. A. Eschmann, E. R. Georgieva, P. Ganguly, P. P. Borbat, M. D. Rappaport, Y. Akdogan, J. H. Freed, J. -E. Shea, and S. Han.
Sci. Rep. 7, 44739 (2017)
Supporting Information
<doi: 10.1038/srep44739>
PMID:
28303942
PMCID:
PMC5356194
Publication #413
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ABSTRACT: The self-assembly of the microtubule associated tau protein into fibrillar cell inclusions is linked to a number of devastating neurodegenerative disorders collectively known as tauopathies. The mechanism by which tau self-assembles into pathological entities is a matter of much debate, largely due to the lack of direct experimental insights into the earliest stages of aggregation. We present pulsed double electron-electron resonance measurements of two key fibril-forming regions of tau, PHF6 and PHF6*, in transient as aggregation happens. By monitoring the end-to-end distance distribution of these segments as a function of aggregation time, we show that the PHF6(*) regions dramatically extend to distances commensurate with extended β-strand structures within the earliest stages of aggregation, well before fibril formation. Combined with simulations, our experiments show that the extended β-strand conformational state of PHF6(*) is readily populated under aggregating conditions, constituting a defining signature of aggregation-prone tau, and as such, a possible target for therapeutic interventions.
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Bacterial Energy Sensor Aer Modulates the Activity of the Chemotaxis Kinase CheA Based on the Redox State of the Flavin Cofactor
D. Samanta, J. Widom, P. P. Borbat, J. H. Freed, and B. R. Crane.
J. Biol. Chem. 291, 25809-25814 (2016)
<doi: 10.1074/jbc.C116.757492>
PMID:
27803157
PMCID:
PMC5207056
Publication #412
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ABSTRACT: Flagellated bacteria modulate their swimming behavior in response to environmental cues through the CheA/CheY signaling pathway. In addition to responding to external chemicals, bacteria also monitor internal conditions that reflect the availability of oxygen, light, and reducing equivalents, in a process termed "energy taxis." In Escherichia coli, the transmembrane receptor Aer is the primary energy sensor for motility. Genetic and physiological data suggest that Aer monitors the electron transport chain through the redox state of its FAD cofactor. However, direct biochemical data correlating FAD redox chemistry with CheA kinase activity have been lacking. Here, we test this hypothesis via functional reconstitution of Aer into nanodiscs. As purified, Aer contains fully oxidized FAD, which can be chemically reduced to the anionic semiquinone (ASQ). Oxidized Aer activates CheA, whereas ASQ Aer reversibly inhibits CheA. Under these conditions, Aer cannot be further reduced to the hydroquinone, in contrast to the proposed Aer signaling model. Pulse ESR spectroscopy of the ASQ corroborates a potential mechanism for signaling in that the resulting distance between the two flavin-binding PAS (Per-Arnt-Sim) domains implies that they tightly sandwich the signal-transducing HAMP domain in the kinase-off state. Aer appears to follow oligomerization patterns observed for related chemoreceptors, as higher loading of Aer dimers into nanodiscs increases kinase activity. These results provide a new methodological platform to study Aer function along with new mechanistic details into its signal transduction process.
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Transport Domain Unlocking Sets the Uptake Rate of an Aspartate Transporter
N. Akyuz, E.R. Georgieva, Z. Zhou, S. Stolzenberg, M.A. Cuendet, G. Khelashvili, R.B. Altman, D.S. Terry, J.H. Freed, H. Weinstein, O. Boudker, and S.C. Blanchard.
Nature 518, 68-73 (2015).
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Transport Domain Unlocking Sets the Uptake Rate of an Aspartate Transporter
N. Akyuz, E.R. Georgieva, Z. Zhou, S. Stolzenberg, M.A. Cuendet, G. Khelashvili, R.B. Altman, D.S. Terry, J.H. Freed, H. Weinstein, O. Boudker, and S.C. Blanchard.
Nature 518, 68-73 (2015).
<doi: 10.1038/nature14158>
PMID:
25652997
PMCID:
PMC4351760
Publication #392
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Aggregation Propensities of Superoxide Dismutase G93 Hotspot Mutants Mirror ALS Clinical Phenotypes
A.J. Pratt, D.S. Shin, G.E. Merz, R.P. Rambo, W.A. Lancaster, K.N. Dyer, P.P. Borbat, F.L. Poole II, M.W.W. Adams, J.H. Freed, B.R. Crane, J.A. Tainer, and E.D. Getzoff.
Proc. Natl. Acad. Sci. U. S. A. 111, E4568-E4576 (2014).
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Aggregation Propensities of Superoxide Dismutase G93 Hotspot Mutants Mirror ALS Clinical Phenotypes
A.J. Pratt, D.S. Shin, G.E. Merz, R.P. Rambo, W.A. Lancaster, K.N. Dyer, P.P. Borbat, F.L. Poole II, M.W.W. Adams, J.H. Freed, B.R. Crane, J.A. Tainer, and E.D. Getzoff.
Proc. Natl. Acad. Sci. U. S. A. 111, E4568-E4576 (2014).
<doi: 10.1073/pnas.1308531111>
PMID:
25316790
PMCID:
PMC4217430
Publication #389
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Pulse Dipolar Electron Spin Resonance: Distance Measurements
P.P. Borbat and J.H. Freed.
In Structural Information from Spin-Labels and Intrinsic Paramagnetic Centers in the Biosciences. Structure and Bonding. Vol. 152. J. Harmer and C. Timmel, Eds.
Springer: Heidelberg, Germany; New York, USA, 2013; pp. 1-82.
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Pulse Dipolar Electron Spin Resonance: Distance Measurements
P.P. Borbat and J.H. Freed.
In Structural Information from Spin-Labels and Intrinsic Paramagnetic Centers in the Biosciences. Structure and Bonding. Vol. 152. J. Harmer and C. Timmel, Eds.
Springer: Heidelberg, Germany; New York, USA, 2013; pp. 1-82.
<doi: 10.1007/430_2012_82>
PMID: [None - Book] PMCID:
[None - Book]
Publication #383
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Membrane Fluidity
B. Dzikovski and J.H. Freed.
In Encyc. Biophys.; G.C.K. Roberts, Ed.
Springer Verlag: Berlin, Germany, 2013; pp 1440-1446.
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Membrane Fluidity
B. Dzikovski and J.H. Freed.
In Encyc. Biophys.; G.C.K. Roberts, Ed.
Springer Verlag: Berlin, Germany, 2013; pp 1440-1446.
<doi: 10.1007/978-3-642-16712-6_546>
PMID: [None - Book] PMCID:
[None - Book]
Publication #381
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The Internal Dynamics of Mini c TAR DNA Probed by Electron Paramagnetic Resonance of Nitroxide Spin-Labels at the Lower Stem, the Loop, and the Bulge
Y. Sun, Z. Zhang, V.M. Grigoryants, W.K. Myers, F. Liu, and K. Earle, J.H. Freed, C.P. Scholes
Biochemistry 51, 8530-8541 (2012).
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The Internal Dynamics of Mini c TAR DNA Probed by Electron Paramagnetic Resonance of Nitroxide Spin-Labels at the Lower Stem, the Loop, and the Bulge
Y. Sun, Z. Zhang, V.M. Grigoryants, W.K. Myers, F. Liu, and K. Earle, J.H. Freed, C.P. Scholes
Biochemistry 51, 8530-8541 (2012).
Supporting Information
<doi: 10.1021/bi301058q>
PMID:
23009298
PMCID:
PMC3549007
Publication #379
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Molecular Motions
S.K. Misra and J.H. Freed.
In Multifrequency Electron Paramagnetic Resonance, S.K. Misra, Ed.
Wiley-VCH: New York, 2011; Chapter 11, pp. 497-544.
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Molecular Motions
S.K. Misra and J.H. Freed.
In Multifrequency Electron Paramagnetic Resonance, S.K. Misra, Ed.
Wiley-VCH: New York, 2011; Chapter 11, pp. 497-544.
<doi: 10.1002/9783527633531>
PMID: [None - Book] PMCID:
[None - Book]
Publication #367
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Diphthamide Biosynthesis Requires an Organic Radical Generated by an Iron-Sulphur Enzyme
Y. Zhang, X. Zhu, A.T. Torelli, M. Lee, B. Dzikovski, R.M. Koralewski, E. Wang, J.H. Freed, C. Krebs, S.E. Ealick, and H. Lin.
Nature, 465, 891-896 (2010).
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Diphthamide Biosynthesis Requires an Organic Radical Generated by an Iron-Sulphur Enzyme
Y. Zhang, X. Zhu, A.T. Torelli, M. Lee, B. Dzikovski, R.M. Koralewski, E. Wang, J.H. Freed, C. Krebs, S.E. Ealick, and H. Lin.
Nature, 465, 891-896 (2010).
Supporting Information
<doi: 10.1038/nature09138>
PMID:
20559380
PMCID:
PMC3006227
Publication #351
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Molecular-Scale Force Measurement in a Coiled-Coil Peptide Dimer by Electron Spin Resonance
S.V. Gullà, G. Sharma, P.Borbat, J.H. Freed, H. Ghimire, M.R. Benedikt, N.L. Holt, G.A. Lorigan, K. Rege, C. Mavroidis, and D.E. Budil.
J. Am. Chem. Soc. 131, 5374-5375 (2009).
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Molecular-Scale Force Measurement in a Coiled-Coil Peptide Dimer by Electron Spin Resonance
S.V. Gullà, G. Sharma, P.Borbat, J.H. Freed, H. Ghimire, M.R. Benedikt, N.L. Holt, G.A. Lorigan, K. Rege, C. Mavroidis, and D.E. Budil.
J. Am. Chem. Soc. 131, 5374-5375 (2009).
Supporting Information
<doi: 10.1021/ja900230w>
PMID:
19331323
PMCID:
PMC2888838
Publication #346
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Membrane Fluidity
B. Dzikovski and J.H. Freed.
In Wiley Encyclopedia of Chemical Biology; T. P. Begley and B. A. Baird, Eds.
Wiley; Hoboken, NJ, USA, 2009; Chapter 2, pp 728-741.
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Membrane Fluidity
B. Dzikovski and J.H. Freed.
In Wiley Encyclopedia of Chemical Biology; T. P. Begley and B. A. Baird, Eds.
Wiley; Hoboken, NJ, USA, 2009; Chapter 2, pp 728-741.
PMID: [None - Book] PMCID:
[None - Book]
Publication #340
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High-Frequency ESR at ACERT
K.A. Earle, B. Dzikovski, W. Hofbauer, J.K. Moscicki and J.H. Freed.
Magn. Reson. Chem. 43, S256-S266 (2005).
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ESR and Molecular Dynamics
J.H. Freed.
In Biomedical EPR, Part B: Methodology, Instrumentation and Dynamics, S.S. Eaton, G.R. Eaton, and L.J. Berliner, Eds.
Biological Magnetic Resonance 24, Chapter 9, pp. 239-268 (2005). (Kluwer, NY).
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ESR and Molecular Dynamics
J.H. Freed.
In Biomedical EPR, Part B: Methodology, Instrumentation and Dynamics, S.S. Eaton, G.R. Eaton, and L.J. Berliner, Eds.
Biological Magnetic Resonance 24, Chapter 9, pp. 239-268 (2005). (Kluwer, NY).
<doi: 10.1007/0-306-48533-8_9>
Publication #306
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Enrichment of Endoplasmic Reticulum with Cholesterol Inhibits Sarcoplasmic-Endoplasmic Reticulum Calcium ATPase-2b Activity in Parallel with Increased Order of Membrane Lipids
Y. Li, M. Ge, L. Ciani, G. Kuriakose, E.J. Westover, M. Dura, D.F. Covey, J.H. Freed, F.R. Maxfield, J. Lytton, and I. Tabas.
J. Biol. Chem. 279, 37030-37039 (2004).
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Enrichment of Endoplasmic Reticulum with Cholesterol Inhibits Sarcoplasmic-Endoplasmic Reticulum Calcium ATPase-2b Activity in Parallel with Increased Order of Membrane Lipids
Y. Li, M. Ge, L. Ciani, G. Kuriakose, E.J. Westover, M. Dura, D.F. Covey, J.H. Freed, F.R. Maxfield, J. Lytton, and I. Tabas.
J. Biol. Chem. 279, 37030-37039 (2004).
<doi: 10.1074/jbc.M405195200>
PMID:
15215242
Publication #299
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Double Quantum ESR and Distance Measurements
P.P. Borbat and J.H. Freed.
In Distance Measurements in Biological Systems by EPR, L.J. Berliner, S.S. Eaton, and G.R, Eaton, Eds.
Biological Magnetic Resonance 19, Chapter 9, pp. 383-459 (2000). (Kluwer, NY).
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Study of Motional Dynamics in Complex Fluids by Very High-Field, Very High-Frequency EPR (VHF-EPR)
A.K. Hassan, J. van Tol, A.L. Maniero, L.C. Brunel, K.A. Earle, and J.H. Freed.
In Physical Phenomena at High Magnetic Fields III, Z. Fisk, L. Gor'kov, J.R. Schrieffer, Eds.
World Scientific NJ, 453-456 (1999)
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Study of Motional Dynamics in Complex Fluids by Very High-Field, Very High-Frequency EPR (VHF-EPR)
A.K. Hassan, J. van Tol, A.L. Maniero, L.C. Brunel, K.A. Earle, and J.H. Freed.
In Physical Phenomena at High Magnetic Fields III, Z. Fisk, L. Gor'kov, J.R. Schrieffer, Eds.
World Scientific NJ, 453-456 (1999)
<doi: 10.1142/3945>
Publication #263
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ESR Studies of Nitrogen Oxides Adsorbed on Zeolite Catalysts: Analysis of Motional Dynamics
H. Yahiro, M. Shiotani, J.H. Freed, M. Lindgren, and A. Lund.
In Catalysis by Microporous Materials, H.K. Beyer, H.G. Karge, I. Kiricsi, and J.B. Nagy, Eds.
Studies in Surface Science and Catalysis Volume 94, pp 673-680 (1995).
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ESR Studies of Nitrogen Oxides Adsorbed on Zeolite Catalysts: Analysis of Motional Dynamics
H. Yahiro, M. Shiotani, J.H. Freed, M. Lindgren, and A. Lund.
In Catalysis by Microporous Materials, H.K. Beyer, H.G. Karge, I. Kiricsi, and J.B. Nagy, Eds.
Studies in Surface Science and Catalysis Volume 94, pp 673-680 (1995).
<doi: 10.1016/S0167-2991(06)81283-8>
Publication #234
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Primary Events in Photosynthesis: Problems, Speculations, Controversies and Future Trends
M. Bixon, J. Fajer, G. Feher, J.H. Freed, D. Gamliel, A.J. Hoff, H. Levanon, K. Möbius, R. Nechushtai, J.R. Norris, A. Scherz, J.L. Sessler, and D. Stehlik.
Isr. J. Chem. 32, 471-482 (1992).
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Primary Events in Photosynthesis: Problems, Speculations, Controversies and Future Trends
M. Bixon, J. Fajer, G. Feher, J.H. Freed, D. Gamliel, A.J. Hoff, H. Levanon, K. Möbius, R. Nechushtai, J.R. Norris, A. Scherz, J.L. Sessler, and D. Stehlik.
Isr. J. Chem. 32, 471-482 (1992).
<doi: 10.1002/ijch.199200050>
Publication #209
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ESR and Slow Motions in Liquid Crystals
J.H. Freed, A. Nayeem, and S.B. Rananavare.
In The Molecular Dynamics of Liquid Crystals; G. Luckhurst and C. Veracini, Eds.
Kluwer: Dordrecht, Netherlands, 1994; Chapter 15, pp. 365-402.
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Two-Dimensional Electron Spin Resonance
J. Gorcester, G.L. Millhauser, and J.H. Freed.
In Modern Pulsed and Continuous-Wave Electron Spin Resonance, L. Kevan and M. Bowman, Eds.
Wiley: NY, 1990; Chapter 3, pp. 119-194.
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Two-Dimensional Electron Spin Resonance
J. Gorcester, G.L. Millhauser, and J.H. Freed.
In Modern Pulsed and Continuous-Wave Electron Spin Resonance, L. Kevan and M. Bowman, Eds.
Wiley: NY, 1990; Chapter 3, pp. 119-194.
Publication #182
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Two Dimensional and Fourier Transform ESR
J. Gorcester, G.L. Millhauser, and J.H. Freed.
In Advanced EPR: Applications in Biology and Biochemistry, A.J. Hoff, Ed.
Elsevier: Amsterdam, 1989; Chapter 8, pp. 177-242.
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Spin Relaxation and Motional Dynamics
D.J. Schneider and J.H. Freed.
In Lasers, Molecules, and Methods, J.O. Hirschfelder, R.E. Wyatt, and R.D. Coalson, Eds.
Adv. Chem. Phys. 73, pp. 387-528 (1989).
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Gyromagnetic Ratio
J.H. Freed.
In Encyclopedia of Physics: G.L. Trigg and R. Lerner, Eds.
Addison-Wesley: New York, 1980; pp. 376-377.
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Gyromagnetic Ratio
J.H. Freed.
In Encyclopedia of Physics: G.L. Trigg and R. Lerner, Eds.
Addison-Wesley: New York, 1980; pp. 376-377.
Publication #65
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