Prediction and Selection of Spin Label Rotamers for High-Resolution Insight into Protein Structures by DEER Spectroscopy

Abstract

Double electron-electron resonance (DEER) spectroscopy is a powerful technique used to obtain structural information about globular and membrane proteins, as well as protein complexes. Typically, the paramagnetic (i.e., “DEER-active”) nitroxide groups of spin labels are linked to cysteine residues of proteins allowing for distance measurements between two spin-labelled cysteine residues. However, free rotation of bonds between the nitroxide groups and the cysteine's α-carbon atoms complicates the deduction of precise distances within the peptide backbone. In this study, we used an intrinsically immobilized imidazoline nitroxide side chain, also referred to as V1, to minimize this effect. Using DEER spectroscopy and X-ray crystallography, we determined some of the most frequently occurring V1 rotamers on various sites of the model protein T4 lysozyme. Finally, we assessed how these rotamers are influenced by either interactions with particular neighbouring amino acid residues or by tertiary steric contacts. In conclusion, this work not only helps to better interpret the data from DEER spectroscopy experiments, but also better prepares the scientist to select more suitable sites for spin labelling.