Application Of Multifrequency Electron Spin Resonance Spectroscopy To Study Dynamics Of T4 Lysozyme

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An extensive set of ESR spectra was obtained over a wide range of frequencies (9, 95, 170 and 240 GHz) and temperatures (2 to 32oC) to explore the dynamic modes of nitroxide-labeled T4 lysozyme in solution. A commonly used nitroxide side chain (R1), or a methylated analogue with hindered internal motion (R2), was substituted for the native side chain at solvent-exposed helical sites, 72 or 131. The spectra at all four frequencies were simultaneously fit with the slowly relaxing local structure (SRLS) model. Good fits were achieved at all the temperatures. Two principle dynamic modes are included in the SRLS model: the global tumbling of the protein and the internal motion consisting of backbone fluctuations and side chain isomerizations. Three distinct spectral components were required for R1 and two for R2 to account for the spectra at all temperatures. One is a highly ordered and slow motional component, which is observed in the spectra of both R1 and R2; it may correspond to conformers stabilized by interaction with the protein surface. The fraction of this component decreases with increasing temperature, and is more populated in the R2 spectra, possibly arising from stronger interaction of the nitroxide ring with the protein surface due to the additional methyl group. The other two components of R1 and the second component of R2, are characterized by fast anisotropic diffusion and relatively low ordering, most likely corresponding to conformers having little or no interactions with nearby residues. Comparison of the motion of R1 and R2 at sites 72 and 131 makes it unclear whether backbone dynamics or differences in side chain internal motion is the major contribution to the spectral differences at these sites. Ficoll of different concentrations was added to increase the solution viscosity, thereby slowing down the global tumbling of the protein. A significant effect of Ficoll on the internal motion of an immobilized component was apparent in R2 but not in R1. The ability of such multifrequency studies to separate the effects of faster internal modes of motion from slower overall motions is clearly demonstrated, and its utility in future studies is considered.

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