Multidimensional Solid-State NMR of Functional Chemotaxis Receptor Signaling Complexes

Abstract

Bacterial chemotaxis receptors serve as an ideal model system for elucidating molecular mechanisms of transmembrane signaling, due to the experimental accessibility and established biochemical characterization of these proteins. A gap in the structural understanding of bacterial chemotaxis is present between electron microscopy of the intact arrays of receptor complexes found in cells and high-resolution crystal structures of individual proteins and truncated complexes. To bridge this gap we have initiated multidimensional solid-state NMR spectroscopy studies of a kinase-active ternary complex composed of the soluble 30 kDa cytoplasmic fragment (CF) of the aspartate chemoreceptor, the 70 kDa histidine kinase CheA, and the 18 kDa adaptor protein CheW. We prepared uniformly 13C & 15N labeled CF and obtained both homonuclear and heteronuclear correlation spectra of frozen and unfrozen complexes.The initial data indicates that such experiments are feasible on this functional multi-protein complex. A number of narrow resonances with sub-ppm linewidths are observed in spectra of both frozen and unfrozen samples, and both states exhibit sufficient signal-to-noise for acquiring multidimensional spectra. Overall, the spectra demonstrate the strongly overlapping resonances expected for an alpha-helical 30 kDa protein, which suggests selective labeling for future measurements of structural constraints. For these samples containing uniformly labeled receptor fragment, comparison with predicted chemical shift spectra are being used to test the current structural model. Comparisons of frozen and unfrozen spectra are also being used to assess the presence of static disorder or dynamics in the side chain and backbone resonances. The goal for uniformly labeled receptor samples is to compare spectral properties of both signaling states to gain insight into possible differences in structure and dynamics.This research supported by NIH grant GM085288.