Structural and functional analysis of novel microbial membrane proteins

点击翻译按钮获取中文摘要

Microorganisms contend with a host of environmental threats, ranging from chemical toxins to dynamically changing ionic conditions. In response, microbes have evolved a unique catalog of membrane transporters and signaling proteins. My research program integrates cutting edge approaches in electrophysiology, membrane protein biophysics, x-ray crystallography, and cryo-EM for the molecular and physiological characterization of membrane proteins that contribute to uniquely microbial physiologies. Currently, three major areas of inquiry are 1) molecular mechanisms for membrane export of environmental toxins 2) molecular mechanisms of receptors that sense and integrate information about changing ionic gradients 3) development of new approaches to overcome challenges in structural characterization of small membrane proteins. For the first line of inquiry, we build off our identification and characterization of two previously unannotated microbial physiologies, fluoride and guanidinium export. These ions are common in the microbial milieu and have broad- spectrum inhibitory effects on microbial metabolism. We provided the first identification and mechanistic and structural characterization of bacterial exporters of these toxins. Future efforts will focus on a) determining the molecular mechanism and first structure of fluoride exporters of pathogenic eukaryotic microbes, known as FEX. These studies will provide molecular information that can be applied to inhibitor design, as well as broad- based insight into membrane protein evolution. b) biophysical analysis of guanidinium exporter Gdx. Together with our recent structures, this project will reveal the mechanistic basis for promiscuous substrate recognition and substrate-coupled conformational change, generating key insight into multidrug transport mechanisms more generally. For the second line of research, we will establish molecular mechanisms of signaling proteins that enact biofilm or virulence programs in response to changing ionic conditions. Our first target is a histidine kinase receptor, KinC, that detects changes in environmental potassium. To understand the biophysical basis for receptor activation, we will evaluate structural ensembles by cryoEM, employ site-directed mutagenesis and in vivo assays for receptor activation, and reconstitute signaling function in lipid vesicles. This work will pioneer biophysical research into ion-gradient-responsive signaling, with implications for pathogenic processes like host colonization and biofilm growth. Our third major research thrust is to develop new approaches to overcome challenges of structural characterization of small membrane proteins. We recently designed a new and efficient approach to generate crystallization chaperones and cryo-EM fiducials, and we will continue to develop this technology in order to make it accessible for as many membrane protein targets and labs as possible. Together, these research activities will generate novel insights into fundamental questions in membrane protein mechanism and microbial membrane physiology and pave the way for the development of novel antibiotics and anti-biofilm agents targeting membrane export and signaling processes.