ACERT continues research on SARS-CoV-2 (SARS-2) structure, in collaboration with other Cornell University laboratories—new results on SARS-2 increased host membrane perturbation found.
In ACERT's efforts to understand the SARS-CoV-2 (SARS-2) structure, especially its Spike protein Fusion Peptide (FP), we and our long-time collaborators, the labs of Gary Whittaker in the Microbiology Dept. of the Vet School and Susan Daniel of Chemical and Biomolecular Engineering, have discovered an important new piece of information. We found results that the SARS-2 FP, like the FPs of the original SARS-CoV, MERS-CoV, and Ebolavirus, has its effectiveness significantly affected by the presence of Ca2+ ions in the local environment1, 2, 3 and the response to Ca2+ is very specific. Our current investigations show that the SARS-2 FP, which differs from the FP of the original SARS by only three residues, is about twice as effective in perturbing membrane ordering of host cells, and thus infecting them. Also, it has a stronger interaction with Ca2+. Since the FP functions to anchor the virus to a host cell, so it can inject its genetic material into the host, knowing that these three residues significantly enhance the infectiousness of SARS-2 suggests a potentially important specific-target therapeutic line of attack against it, as well as therapeutic solutions to block the Ca2+ channel.
In this work we developed an innovative approach to biomedical ESR measurements, wherein instead of investigating the FPs in isolation, we study the FPs in the context of complete S proteins that are pre-assembled in the form of a trimer on native membranes. This is a more "biological scenario" than the isolated FP. Also we take "time-lapse" snapshots of a [membrane/FP] sample so that we could plot the growth of membrane perturbation in real time over the course of minutes. We were able to obtain ESR signals with high fidelity through use of our powerful wavelet-based denoising procedures. (See here for our signal denoising publications.)
Our research is supported by the NIH National Institute of General Medical Sciences (NIGMS), through grants R01GM123779 and P41GM103521. Our present COVID-19 research is a direct progression from our multi-year collaboration with the Whittaker and Daniel groups, building on our prior work on other viruses (SARS-1, MERS, Ebola, Influenza A, etc.)..
SARS-CoV-2 Fusion Peptide Has a Greater Membrane Perturbation Effect than SARS-CoV with Highly Specific Dependence on Ca2+. A. L. Lai and J. H. Freed. A preprint has been posted to bioRxiv, doi = 10.1101/2021.01.04.425297.
1 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)
2 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 Infectious Diseases 6, 250-260 (2020)
3 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. Journal of Virology 94, e00426-20 (2020)
ACERT demonstrates ability to measure chemical exchange processes at microsecond time-scales via 2D-ELDOR at 95GHz.
Chemical exchange processes such as conformational change, protonation/deprotonation, and binding equilibria occur at many timescales. 2D-NMR enables real-time study of millisecond and slower exchange processes. But while 2D-ELDOR (two-dimensional electron-electron double resonance) has had the potential for studying such processes in the nanosecond to microsecond regime, the most common ESR probes such as nitroxides, studied at typical ESR frequency 9 GHz, are unable to resolve exchanging states from each other within their respective line widths. At 95 GHz, however, 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. As the culmination of a several-year upgrade effort to our 95 GHz spectrometer, we recently demonstrated these 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.
Publication: 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. JACS 142, 21368-21381 (web-published, Dec. 11, 2020); doi: 10.1021/jacs.0c09469; PMCID: PMC7810061.
ACERT continues agency-approved essential research on SARS-CoV-2 (SARS-2) structure, in collaboration with other Cornell University laboratories.
Our ACERT center continues its contribution to understanding the SARS-CoV-2 (SARS-2) structure, especially its Spike protein Fusion Peptide (FP) and its envelope E protein. The FP is a critical portion of the viral envelope, allowing the virus to anchor to a host cell and inject its genetic material into the host. We have found preliminary results that the SARS-2 FP, like the FP of the original SARS, Ebola, and some other coronaviruses, has its effectiveness significantly affected by the presence of Ca2+ ions in the local environment.1 This interaction suggests a potentially important therapeutic line of attack against it. All of our research is supported by the NIH National Institute of General Medical Sciences (NIGMS). We are employing Electron Resonance Spectroscopy (ESR/EPR), both continuous-wave and pulsed, to investigate viral protein structure and dynamics. In this, we are continuing a multi-year collaboration with the labs of Gary Whittaker in the Microbiology Dept. of the Vet School and Susan Daniel of Chemical and Biomolecular Engineering.2 In addition to our larger P41 NIH-funded Center grant, we have a second NIH grant to study membrane protein structure and dynamics and their effects on membranes. Our present COVID-19 research is a direct, logical progression of this project to our collaboration with the Whittaker and Daniel groups from other viruses we have been studying (SARS-1, MERS, Ebola, Influenza A, etc.).
1 A.L. Lai, J.K. Millet, S. Daniel, J.H. Freed, G.R. Whittaker, The SARS-CoV fusion peptide forms an extended bipartite fusion platform that perturbs membrane order in a calcium-dependent manner, J. Mol. Biol., 429 (2017), pp. 3875-3892, 10.1016/j.jmb.2017.10.017
2 Researchers seek universal treatments to impede coronavirus, Cornell Chronicle, 2020/04