Structural mechanisms for ABC multidrug transporter selectivity and transport

 描述(由申请人提供)：ABC转运蛋白控制药物、脂类和多肽在人体内的分布，并在多药耐药中发挥关键作用。这项建议旨在了解ABC转运体的整个运输周期，并通过结合X射线结晶学和低温电子显微镜的强大组合方法，确定控制不同ABC转运体如何选择底物的规则。这里概述的方法研究了ABC转运体的机械循环，从底物结合到构象变化，使用冷冻电子显微镜(Cryo-EM)方法，在Tomasiak博士与程一凡博士的合作中，获得了前所未有的ABC转运体的亚纳米分辨率结构。最终目标是建立几种人类转运蛋白的综合模型，以揭示人类药代动力学和药物基因组学的定量模型，从而更清楚地了解这些生物关键分子是如何分布的。候选人Thomas Tomasiak有兴趣利用K99开始独立的职业生涯，编织几个生物学学科和方法来研究药物、脂质和其他疏水化合物的分布。Tomasiak博士希望成为一家领先机构的创新助理教授，为我们在微生物发病机制和癌症中的药物处置和多药耐药性方面的知识做出贡献。这项提议的核心是一个冷冻-EM培训项目，该项目将帮助Tomasiak博士成为该领域的专家，并将其与他在X射线结晶学方面的背景相结合。膜蛋白结晶学和冷冻-EM的结合将使一种强大的杂交方法能够回答有关膜蛋白构象变化和复合体的生物学问题，这两种技术单独使用都是困难的。拟议研究的一部分是开发用于验证来自低温EM的结构模型的新工具。这项提议旨在通过将托马斯目前在膜蛋白和X射线结晶学方面的知识与新兴的冷冻电子显微镜领域相结合，来补充他的培训。Tomasiak博士的导师斯特劳德博士和共同导师程博士在各自的领域都是国际公认的，斯特劳德博士支持了许多博士后研究人员过渡到独立。除了联合导师，托马夏克博士还组建了一个由资深科学家组成的顾问委员会，他们将能够帮助指导他的研究，并处于指导他职业生涯的理想位置 晋升到一个独立的职位。

 DESCRIPTION (provided by applicant): ABC transporters govern distribution of drugs, lipids, and peptides throughout the human body and play a critical role in multidrug resistance. This proposal aims to understand the entire transport cycle of an ABC transporter and identify rules governing how different ABC transporters select substrates by using a powerful combination approach combining x-ray crystallography and cryo electron microscopy. The approach outlined here examines the mechanistic cycle of an ABC transporter, from substrate binding to conformational changes using a cryo electron microscopy (cryo-EM) approach that has yielded an unprecedented subnanometer resolution structure of an ABC transporter in a collaboration Dr. Tomasiak was part of with Dr. Yifan Cheng. The ultimate goal is a comprehensive model of several human transporters to reveal a quantitative model of human pharmacokinetics and pharmacogenomics that will give a clearer understanding of how these biologically critical molecules are distributed. The candidate, Thomas Tomasiak, has an interest to use the K99 to launch an independent career weaving several biological disciplines and approaches to studying distribution of drugs, lipids, and other hydrophobic compounds. Dr. Tomasiak hopes to become an innovate assistant professor at a leading institution, contributing to our knowledge of drug disposition and multidrug resistance in microbial pathogenesis and cancer. This proposal is centered on a training program in cryo-EM that will help Dr. Tomasiak become an expert in this field and combine it with his background in x-ray crystallography. The combination of membrane protein crystallography and cryo-EM will make a powerful hybrid approach to answer biological questions concerning conformational state changes and complexes in membrane proteins difficult for either technique alone. Part of the proposed research is in development of new tools for the validation of structural models from cryo-EM. The proposal is designed to supplement Thomas's training by integrating his current knowledge of membrane proteins and x-ray crystallography with the burgeoning field of cryo-electron microscopy. Dr. Tomasiak's mentor, Dr. Stroud, and co-mentor, Dr. Cheng are internationally recognized in their fields and Dr. Stroud has supported numerous postdoctoral researchers into the transition to independence. In addition to the co-mentors, Dr. Tomasiak has assembled an advisory committee of senior scientists who will be able to help guide his research and are ideally situated to guide his career progression to an independent position.