Structural and functional analysis of novel microbial membrane proteins

微生物与一系列环境威胁作斗争，从化学毒素到动态的 改变离子条件。作为回应，微生物已经进化出一个独特的膜转运蛋白目录， 信号蛋白我的研究计划整合了电生理学，膜 蛋白质生物物理学，x射线晶体学和冷冻电镜的分子和生理特性， 膜蛋白，有助于独特的微生物生理。目前，三大调查领域 是1）环境毒素的膜输出的分子机制2）环境毒素的膜输出的分子机制 感受和整合有关变化的离子梯度的信息的受体3）新的 克服小膜蛋白结构表征挑战的方法。面向基层一线 调查，我们建立了我们的鉴定和表征两个以前未注释的微生物 生理学、氟化物和胍出口。这些离子在微生物环境中很常见，具有广泛的- 对微生物代谢的光谱抑制作用。我们提供了第一个识别和机械， 这些毒素的细菌输出者的结构表征。未来的工作将集中在a）确定 致病性真核微生物的氟输出分子机制和第一结构， 外汇。这些研究将提供可应用于抑制剂设计的分子信息，以及广泛的 基于对膜蛋白进化的洞察。B）胍输出体Gdx的生物物理分析。一起 利用我们最近的结构，这个项目将揭示混杂底物识别的机制基础 和底物偶联的构象变化，产生对多药物转运机制的关键见解 更普遍地说。对于第二条研究路线，我们将建立信号蛋白的分子机制 其响应于变化的离子条件而制定生物膜或毒力程序。我们的第一个目标是组氨酸 激酶受体KinC，检测环境钾的变化。为了理解生物物理学基础 对于受体活化，我们将通过cryoEM评估结构集合，采用定点诱变， 用于受体活化的体内测定，并重建脂质囊泡中的信号传导功能。这项工作将开创 离子梯度响应信号的生物物理研究，对致病过程的影响，如 宿主定植和生物膜生长。我们的第三个主要研究重点是开发新的方法， 克服了小膜蛋白结构表征的挑战。我们最近设计了一个新的 和有效的方法来产生结晶分子伴侣和冷冻EM基准，我们将继续 开发这项技术，使其可用于尽可能多的膜蛋白靶点和实验室， 可能总之，这些研究活动将产生新的见解的基本问题， 膜蛋白机制和微生物膜生理学的研究，为膜蛋白的发展铺平道路 靶向膜输出和信号传导过程的新型抗生素和抗生物膜剂。

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.