Pulsed ESR dipolar spectroscopy for distance measurements in immobilized spin labeled proteins in liquid solution.
Pulsed electron spin resonance (ESR) dipolar spectroscopy (PDS) in combination with site-directed spin labeling is unique in providing nanometer-range distances and 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. In this preliminary study, 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.
Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90095, USA.
Zhongyu
Z
Yang
Yangping
Y
Liu
Peter
P
Borbat
Jay L
JL
Zweier
Jack H
JH
Freed
Wayne L
WL
Hubbell
D017122:Bacteriophage T4 / Q000201:enzymology*; D003545:Cysteine / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D055767:Immobilized Proteins / Q000737:chemistry*; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry*; D012996:Solutions; D013113:Spin Labels / Q000138:chemical synthesis*; D014867:Water / Q000737:chemistry
D055767:Immobilized Proteins; D012996:Solutions; D013113:Spin Labels; D014867:Water; D009113:Muramidase; D003545:Cysteine