Molecular motion of spin labeled side chains in alpha-helices: Analysis byvariation of side chain structure

Two Single cysteine substitution mutants at helix surface sites in T4 lysoz yme (D72C and V131C) have been modified with a series of nitroxide methanet hiosulfonate reagents to investigate the structural and dynamical origins o f their electron paramagnetic resonance spectra. The novel reagents include 4-substituted derivatives of either the pyrroline or pyrrolidine series of nitroxides. The spectral line shapes were analyzed as a function of side c hain structure and temperature using a simulation method with a single orde r parameter and diffusion rates about three orthogonal axes as parameters. Taken together, the results provide strong support for an anisotropic motio nal model of the side chain, which was previously proposed from qualitative features of the spectra and crystal structures of spin labeled T4 lysozyme . Site-specific differences in apparent order parameter are interpreted in terms of backbone dynamics modes with characteristic correlation times in t he nanosecond or faster time scale. The saturated 4-substituted pyrrolidine nitroxides are shown to be a suitable template for novel "functionalized" side chains designed to mimic salient features of the native side chains th ey replace.