X-ray structures of nitroxide side chains in proteins: A basis for interpreting distance measurements and dynamic studies by electron paramagnetic resonance

Site-directed spin labeling (SDSL) is an exceptional technique for characterizing protein structure and dynamics by Electron Paramagnetic Resonance (EPR). In SDSL, a unique cysteine residue is introduced into a recombinantly expressed protein via site-directed mutagenesis, then reacted with a sulfhydryl specific spin labeling reagent to generate a spin label side chain that acts as a local reporter of dynamic information. The R1 spin label (R1 = [-CH2-S-S-CH 2-3([2,2,5,5, tetramethylpyrroline-1-oxyl]) is extensively used in SDSL studies, and simulations of EPR spectra from a database of spin labeled proteins suggest that the R1 side chain (R1 = [-CH2-S-S-CH2-3([2,2,5,5, tetramethylpyrroline-1-oxyl]) exhibits a limited number of fundamentally different dynamic modes (disordered, weakly ordered, strongly ordered and immobilized).;In order to understand the structural determinants of each different mode, five crystal structures of T4 lysozyme (T4L) bearing the R1 side chain have been determined (81R1, 82R1, 130R1, 131R1, and 150R1) and the rotameric states identified. The structural basis for weakly ordered motion (derived from 131R1 and 82R1) is apparently an interaction of the Sdelta atom with the protein backbone. This interaction restricts isomerizations about the first two dihedral angles (X1 and X2), giving rise to two defined rotamers [(-60°, -60°) and (180°, 60°)], making them an ideal choice for distance determinations or detection of local nanosecond backbone motions.;Strongly ordered motions arise from interactions of the nitroxide with the tertiary structure (81R1 and 150R1) Together with 130R1 and previous structural studies, these structures demonstrate that the R1 side chain results in little perturbation of the native protein fold and adopt well defined rotamers [(180°, 60°), (180°, -60°) and (-60°, 180°)].;To explore the structural nature of the restricted motions at helix surface sites, X-ray crystal structures of T4L spin labeled with a 4-methyl, 4-phenyl, and 4-bromo, and a novel cross-linking derivative of R1 were determined. The structures suggest that the 4-methyl and 4-bromo derivatives are hindered internally, while the motions of the 4-phenyl derivative are restricted by interaction with the protein, supporting their use in detection of local backbone modes by EPR. The cross-linked nitroxide might be useful for detecting axial rocking modes by EPR.