Motion of spin-labeled side chains in T4 lysozyme: Effect of side chain structure

Previous studies have shown that the mobility of nitroxide side chains in a protein, inferred from the electron paramagnetic resonance (EPR) spectra, can be used to classify particular sites as helix surface sites, tertiary c ontact sites, buried sites, or loop sites. In addition, the sequence depend ence of mobility can identify regular secondary structure. However, in the most widely used side chain, an apparent interaction of the nitroxide ring with the protein at some helix surface sites gives rise to EPR spectra dege nerate with those at tertiary contact sites. In the present study, we use s elected sites in T4 lysozyme to evaluate novel nitroxide side chains design ed to resolve this degeneracy. The results indicate that the reagent 3-(met hanesulfonylthiomethyl)-2,2,5,5-tetramethylpyrrolidin-1-yloxy reacts with c ysteine to give a nitroxide side chain that has a high contrast in mobility between helix surface and tertiary contact sites, effectively resolving th e degeneracy. The reagent 3-(iodomercuriomethyl)-2,2,5,5-tetramethyl-2,5-di hydro-1H-pyrrol-1-yloxy reacts with cysteine to provide a mercury-linked ni troxide that also shows reduced interaction with the protein at most helix surface sites. Thus, these new side chains may be the preferred choices for structure determination using site-directed spin labeling.