1 research outputs found
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