Room Temperature Electron Spin Resonance Distance Measurements in T4 Lysozyme using Trityl-Based Spin Labels

Abstract

Pulsed Electron Spin Resonance (ESR) spectroscopy in combination with site-directed spin labeling is unique in providing nanometer- range distances and distributions in biological systems. Distance constraints are valuable for determining structure and structural changes, while distance distributions reflect structural heterogeneity. To date, most of the pulsed ESR techniques require 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 at physiological temperatures, we are exploring a trityl-based spin label with a relatively long relaxation time where the protein is immobilized by attachment to a solid support. In this preliminary study, trityl radicals were attached via disulfide linkages to substituted cysteine residues at positions 65 and 80 or at positions 65 and 76 in T4 lysozyme (T4L) immobilized on Sepharose. Double quantum coherence (DQC) was applied to the two spin-labeled double mutants at room temperature. Distances extracted from DQC show a 2.2 nm and a 1.8 nm spin-spin distance, respectively, close to those expected from models of the trityl label in the T4L structure. Narrow distribution of distances was observed in each case, indicating that the trityl spin label is relatively rigid. The results show that, with the use of trityl spin label and DQC, structural constraints can be obtained to study protein structure/dynamics at physiological temperature. Furthermore, as we show, trityl labels enable micromolar sensitivity DQC distance measurements in the temperature range of 60-100 K, since their distant methyl groups only weakly contribute to the relaxation of electron spins. As an additional advantage less intense microwave pulses are needed as compared to nitroxides.