DETERMINATION OF THE THREE-DIMENSIONAL STRUCTURE OF A TRANSMEMBRANE CHEMORECEPTOR BY CRYO-ELECTRON MICROSCOPY
Narahari Akkaladevi; Tommi A. White and Gerald L. Hazelbauer
Transmembrane chemoreceptors are central components of the highly conserved signaling system that mediates bacterial chemotaxis, movement toward favorable environments and away from unfavorable ones. Among the vast number of sensory systems in biology, the bacterial chemotaxis system is one of the very best characterized at the molecular and mechanistic level. There are three-dimensional structures for many protein components of this complex system but none for an intact, transmembrane chemoreceptor. Thus we cannot answer fundamental mechanistic questions about conformational transmembrane and allosteric signaling within this key component. Attempts over several decades by multiple research groups to determine the three-dimensional structures of intact, functional chemoreceptors by X-ray crystallography were unsuccessful. These failures likely reflected the flexible and dynamic nature of these trans-membrane receptors as well as the disrupted and inactive state of detergent-solubilized chemoreceptors. Our laboratory solved the second issue by utilizing Nanodiscs, small (~10 nm) plugs of lipid bilayer rendered water-soluble by enclosure in a belt formed by a small, amphipathic protein (Boldog et al. 2006). Recently our lab also analyzed molecular shapes of intact homodimers of nanodisc inserted Escherichia coli aspartate receptor Tar by negative stain electron microscopy and identified the presence of two flexible hinges in the extended, rod-like cytoplasmic domain (Akkaladevi et al. communicated). We are now proceeding to optimize preparation and manipulation of Nanodisc-inserted chemoreceptor dimers for structural analysis by high-resolution cryo-electron microscopy. Our initial trial on cryo-EM sample preparation, a few images show nanodisc inserted Tar (Fig. 1a) with reasonably good contrast. About 100 particles picked and performed reference free 2D average classification to increase signal to noise ratio. One of the good 2D class averages show clearly nanodisc and receptor density (Fig. 1c).