A multifrequency electron spin resonance study of T4 lysozyme dynamics using the slowly relaxing local structure model

Electron spin resonance (ESR) spectra were obtained at 250 and 9 GHz for nitroxide-labeled mutants of the protein T4 lysozyme in aqueous solution over a range of temperatures from 2 to 37.5 °C. Two mutants labeled at sites 72 and 131 were studied and compared. The mutant sites are solvent exposed and free of tertiary interactions with other side chains, but the former is at the center of a 5 turn helix, whereas the latter site is on a small two and a half turn helix. The 250 GHz ESR spectra, because of their “fast time scale”, are rather insensitive to the slow overall tumbling motion of the protein. Thus, they are qualitatively different for the two mutants, implying that there are different local dynamics at the two sites. The 9 GHz spectra, which are significantly affected by the overall tumbling and are less sensitive to the internal dynamics, do not show such marked differences between the two sites. The 250 and 9 GHz spectra for each mutant and temperature were simultaneously fit to the slowly relax...