SRLS APPROACH FOR STUDIES ON ORIENTED MEMBRANE PROTEINS BY SOLID-STATE NMR

Cornell University, Ithaca NY

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. In solid-state NMR of oriented membrane proteins, the traditional method of NMR spin relaxation analysis, which assumes predominantly fast isotropic motions, does not extract adequately and fully the information inherent to the experimental data. Here, the primary issue is how to address the great complexity of protein dynamics, including the global and restricted local motions. In general, in oriented membrane proteins one should expect highly constrained local motions of the loop domains and the backbone superimposed onto the overall uniaxial rotational diffusion of the protein (e.g. of the type observed in magnetically aligned bicelles. The unique SRLS approach developed by Freed and co-workers accounts most generally for the relationship between the global and local motions, for the local ordering, and for the relevant magnetic interactions. The implementation of the model for the 2D-ELDOR experiment is directly generalizable to solid-state NMR pulse sequences, which include switching on the transverse B1 field with defined pulse phases and lengths for prolonged periods of time during the evolution. Moreover, the features of the SRLS model allow one to include torsional motions of the protein within the membrane's restoring potential, as well as the effects of collective fluctuations of the membrane on the protein dynamics. We will adapt the SRLS model to solid-state NMR spectroscopy of oriented membrane proteins with the purpose of extracting the dynamic and structural information from the analyses of spectral lineshapes and cross-polarization build-up rates.