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Pulsed ESR dipolar spectroscopy (PDS) in combination with site-directed spin labeling is unique in providing nanometer- range distances and their distributions in biological systems. To date, most of the pulsed ESR techniques require frozen solutions at cryogenic temperatures to reduce the rapid electron spin relaxation rate and to prevent averaging of electron-electron dipolar interaction due to the rapid molecular tumbling. To enable measurements in liquid solution, we are exploring a triarylmethyl (TAM)-based spin label with a relatively long relaxation time where the protein is immobilized by attachment to a solid support. TAM radicals were attached via disulfide linkages to substituted cysteine residues at positions 65 and 80 or 65 and 76 in T4 lysozyme immobilized on Sepharose. Interspin distances determined using double quantum coherence (DQC) in solution are close to those expected from models, and the narrow distance distribution in each case indicates that the TAM-based spin label is relatively localized. The DQC method is ideal for a single narrow resonance line, such as that of the TAM spin label. Shown in the Figure below, on the top: (A) reaction of TAM-based spin labeling reagent (CT02-TP) with a protein SH. (B) shows immobilization of a protein using CNBr sepharose. Shown on the bottom: DQC evolution of TAM-labeled 65/76 immobilized on sepharose at 4°C and distance distribution derived from it. Publication: Z. Yang, Y. Liu, P. Borbat, J.L. Zweier, J.H. Freed, and W.L. Hubbell, J. Am. Chem. Soc., 134, 9950-9952 (2012); PMC3409244. |
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Zhongyu Yang (Jules Stein Eye Institute and Departments of Chemistry and Biochemistry, UCLA, CA) Yangping Liu (Center for Biomedical EPR Spectroscopy and Imaging, The Davis Heart and Lung Research Institute, the Division of Cardiovascular Medicine, Department of Internal Medicine, The Ohio State University, Columbus, OH) Peter Borbat (ACERT) Jay L. Zweier (Center for Biomedical EPR Spectroscopy and Imaging, The Davis Heart and Lung Research Institute, the Division of Cardiovascular Medicine, Department of Internal Medicine, The Ohio State University, Columbus, OH) Jack H. Freed (ACERT) Wayne L. Hubbell (Jules Stein Eye Institute and Departments of Chemistry and Biochemistry, UCLA, CA) |
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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. |
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