ACERT Highlights: ACERT's Service and Collaborative Projects

National Institute of General Medical Sciences
	National Biomedical Resource for Advanced ESR Spectroscopy

ACERT's Service and Collaborative Projects

Site-Specific Incorporation of a Cu²⁺ Spin Label into Proteins for Measuring Distances by Pulsed Dipolar Electron Spin Resonance Spectroscopy

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. We demonstrated site-specific incorporation of Cu²⁺ into non-metalloproteins through the use of a genetically encodable non-natural amino acid, 3-pyrazolyltyrosine (PyTyr). We first incorporated PyTyr in cyan fluorescent protein (CFP) to measure Cu²⁺-to-NO distances and examined the effects of solvent conditions on Cu²⁺ binding and protein aggregation. We then applied 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 provided new insights into the conformational landscape of the phosphotransfer complex and they have implications for kinase regulation.

Funding: R01GM066775, R01GM079679, R35GM122535 (BRC); P41GM103521 (JHF); T32GM008267 (GEM); NE-CAT Advanced Photon Source P30GM124165 and S10RR029205.

Publication: J. Phys. Chem. B 122, 9443-9451 (2018); PMCID: PMC6215709.


Targeted Cu²⁺ incorporation into CFP. (A) Cu²⁺ binding amino acid PyTyr shown with Cu²⁺-NO separation in CFP expected from the crystal structure of CFP (PDB: ₃ZTF) modeled with a nitroxide on residue 208. (B) Distance distribution and denoised time-domain spectrum (inset) obtained by Cu²⁺-NO DEER spectroscopy in H₂O-based buffer. The red regions indicate the small uncertainty arising from the SVD of the denoised signal.		A phosphotransfer complex revealed by PyTyr PDS. (A) Schematic of the undocked (top) and docked (bottom) states of P1 relative to the P2:CheY complex. The copper centers of CheY are represented in blue, the nitroxide label in red. (B) Structural model of the docked state based on NMR data on E. coli proteins. (C) Distance distribution for Cu²⁺-NO (P1) with error analysis (red) shows separations indicative of both major undocked and minor docked states. Both CheY Cu²⁺ centers give similar distances to the P1 nitroxide moiety in the docked configuration. Denoised time-domain data displayed as an inset.

Gregory E. Merz, Peter P. Borbat, Alise R. Muok, Madhur Srivastava, David N. Bunck, Jack H. Freed, and Brian R. Crane (Department of Chemistry & Chemical Biology, Cornell University, Ithaca, NY)

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ACERT is supported by grant 1R24GM146107 from the National Institute of General Medical Sciences (NIGMS), part of the National Institutes of Health.

National Biomedical Resource for Advanced ESR Spectroscopy

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