Crystal structures of spin labeled T4 lysozyme mutants: Implications for the interpretation of EPR spectra in terms of structure

High resolution (1.43-1.8 Angstrom) crystal structures and the correspondin g electron paramagnetic resonance (EPR) spect-ra were determined for T4 lys ozyme derivatives with a disulfide-linked nitroxide side chain [-CH2-S-S-CH 2-(3-[2,2,5,5-tetramethyl pyrroline-1-oxyl]) = R1] substituted at solvent-e xposed helix surface sites (Lys65, Arg80, Arg119) or a tertiary contact sit e (Va175). In each case, electron density is clearly resolved for the disul fide group, revealing distinct rotamers of the side chain, defined by the d ihedral angles X-1 and X-2. The electron density associated with the nitrox ide ring in the different mutants is inversely correlated with its mobility determined from the EPR spectrum. Residue 80R1 assumes a single g(+)g(+) c onformation (X-1 = 286, X-2 = 294). Residue 119R1 has two EPR spectral comp onents, apparently corresponding to two rotamers, one similar to that for 8 0R1 and the other in a tg(-) conformation (X-1 = 175, X-2 = 54). The latter state is apparently stabilized by interaction of the disulfide with a Gin at i + 4, a situation also observed at 65R1. R1 residues at helix surface s ite 65 and tertiary contact site 75 make intra- as well as intermolecular c ontacts in the crystal and serve to identify the kind of molecular interact ions possible for the R1 side chain. A single conformation of the entire 75 R1 side chain is stabilized by a variety of interactions with the nitroxide ring, including hydrophobic contacts and two unconventional C-H ... O hydr ogen bonds, one in which the nitroxide acts as a donor (with tyrosine) and the other in which it acts as an acceptor (with phenylalanine). The interac tions revealed in these structures provide an important Link between the dy namics of the R1 side chain, reflected in the EPR spectrum, and local prote in structure. A Library of such interactions will provide a basis for the q uantitative interpretation of EPR spectra in terms of protein structure and dynamics.