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Structural and functional analysis of novel microbial membrane proteins

新型微生物膜蛋白的结构和功能分析

基本信息

批准号：
10621520
负责人：
Randy B. Stockbridge
金额：
$ 46.05万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-08-01 至 2028-07-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10621520
关键词：
Antibiotics Architecture Area Biological Assay Biophysics Carrier Proteins Catalogs Chemicals Coupled Cryoelectron Microscopy Crystallization Development Drug Design Electrophysiology (science)Environment Evolution Fluorides Future Growth Ions Lipids Membrane Membrane Proteins Membrane Transport Proteins Metabolism Microbe Microbial Biofilms Microbial Physiology Molecular Molecular Chaperones Molecular Conformation Pathogenicity Physiological Physiology Potassium Process Receptor Activation Research Research Activity Signal Transduction Signaling Protein Site-Directed Mutagenesis Structure System Technology Therapeutic Toxic Environmental Substances Toxin Vesicle Virulence Work X-Ray Crystallography biophysical analysis design guanidinium host colonization in vivo inhibitor insight microbial microorganism microorganism interaction multidrug transport novel novel strategies programs protein-histidine kinase receptor reconstitution response targeted agent

项目摘要

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.

微生物应对一系列环境威胁，从化学毒素到动态改变离子条件。作为回应，微生物进化出了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氟化物输出体的分子机制和第一结构，称为外汇。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，受体激活的体内测定，并重建脂囊泡中的信号功能。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，膜蛋白机理和微生物膜生理学的研究，为开发靶向膜输出和信号传导过程的新型抗生素和抗生物膜剂。

项目成果

期刊论文数量（15）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Membrane Exporters of Fluoride Ion.

DOI：
10.1146/annurev-biochem-071520-112507
发表时间：
2021-06-20
期刊：
Annual review of biochemistry
影响因子：
16.6
作者：
McIlwain BC;Ruprecht MT;Stockbridge RB
通讯作者：
Stockbridge RB

The application of Poisson distribution statistics in ion channel reconstitution to determine oligomeric architecture.

DOI：
10.1016/bs.mie.2021.02.018
发表时间：
2021
期刊：
Methods in enzymology
影响因子：
0
作者：
通讯作者：

Molecular Mechanisms for Bacterial Potassium Homeostasis.

DOI：
10.1016/j.jmb.2021.166968
发表时间：
2021-08-06
期刊：
JOURNAL OF MOLECULAR BIOLOGY
影响因子：
5.6
作者：
Stautz, Janina;Hellmich, Yvonne;Fuss, Michael F.;Silberberg, Jakob M.;Devlin, Jason R.;Stockbridge, Randy B.;Haenelt, Inga
通讯作者：
Haenelt, Inga

The structural basis of promiscuity in small multidrug resistance transporters.

DOI：
10.1038/s41467-020-19820-8
发表时间：
2020-11-27
期刊：
Nature communications
影响因子：
16.6
作者：
Kermani AA;Macdonald CB;Burata OE;Ben Koff B;Koide A;Denbaum E;Koide S;Stockbridge RB
通讯作者：
Stockbridge RB

The fluoride permeation pathway and anion recognition in Fluc family fluoride channels.