A multifrequency electron spin resonance study of T4 lysozyme dynamics

Electron spin resonance (ESR) spectroscopy at 250 GHz and 9 GHz is utilized to study the dynamics and local structural ordering of a nitroxide-labeled enzyme, T4 lysozyme (EC 3.2.1.17), in aqueous solution from 10 degrees C t o 35 degrees C. Two separate derivatives, labeled at sites 44 and 69, were analyzed. The 250-GHz ESR spectra are well described by a microscopic order ing with macroscopic disordering (MOMD) model, which includes the influence of the tether connecting the probe to the protein. In the faster "time sca le" of the 250-GHz ESR experiment, the overall rotational diffusion rate of the enzyme is too slow to significantly affect the spectrum, whereas for t he 9-GHz ESR spectra, the overall rotational diffusion must be accounted fo r in the analysis. This is accomplished by using a slowly relaxing local st ructure model (SRLS) for the dynamics, wherein the tether motion and the ov erall motion are both included. In this way a simultaneous fit is successfu lly obtained for both the 250-GHz and 9-GHz ESR spectra. Two distinct motio nal/ordering modes of the probe are found for both lysozyme derivatives, in dicating that the tether exists in two distinct conformations on the ESR ti me scale. The probe diffuses more rapidly about an axis perpendicular to it s tether, which may result from fluctuations of the peptide backbone at the point of attachment of the spin probe.