CAREER: Development of an In Cell Footprinting Method for the Analysis of Protein Structure

膜蛋白参与许多重要的细胞功能。尽管膜蛋白具有重要的生物学意义，但它们的研究一直受到纯化这些蛋白的困难的阻碍。由于大多数关于膜蛋白的研究都要求这些蛋白质处于类膜环境中，因此开发了各种膜模拟系统，这增加了另一层复杂性。该项目将开发一种新颖和创新的方法，通过提供一种在活细胞中天然脂质环境中表征膜蛋白的方法，将显著推进膜蛋白领域的知识。该方法将首先使用具有良好特征的细胞收缩蛋白作为模型系统，然后将膜蛋白血管运动蛋白作为跨膜蛋白研究的模型系统。该项目将为了解膜运输的生理学和病理生理学提供一个途径。该项目将为来自传统黑人学院和大学的学生提供为期八周的本科暑期项目培训，以提供STEM研究机会。这些学生将在连续的夏季接受培训，并在最后一年成为导师。该项目还将为博士生和博士后提供培训。该项目侧重于开发细胞内蛋白质足迹方法，结合质谱法来表征细胞内蛋白质相互作用。研究蛋白质-脂质相互作用的一个主要障碍是模拟细胞的膜环境。该项目开发的方法将通过提供一种在天然细胞环境中表征膜蛋白的方法，在膜蛋白的研究中取得重大进展。该方法使用“笼状过氧化氢”，将首先使用具有良好特征的细胞质蛋白作为模型系统进行开发。该方法的参数将被系统地优化，定量基准将被用来确定成功。统计分析将用于验证观察到的蛋白质修饰变化的意义。然后将膜蛋白血管运动素作为跨膜蛋白研究的模型系统。这个项目将导致一种新的分析方法的发展，用于研究活细胞中细胞环境中的蛋白质。该方法将允许研究目前可用的结构方法无法研究的膜蛋白，并为细胞环境中的膜运输提供新的见解。

Membrane proteins are involved in many important cellular functions. Despite their biological importance, studies with membrane proteins have been hampered by difficulties involved in purification of these proteins. Since most studies with membrane proteins require that these proteins to be in a membrane-like environment, a variety of membrane mimicking systems have been developed, which adds another layer of complexity. This project will develop a novel and innovative approach that will significantly advance knowledge in the field of membrane proteins by providing a method that can characterize them in their native lipid environment in living cells. The method will first be developed using a well-characterized cytostolic protein as a model system and then the membrane protein angiomotin will be used as a model system for the study of transmembrane proteins. The project will provide a gateway to understanding the physiology and pathophysiology of membrane trafficking. This project will provide training for students from Historically Black Colleges and Universities in an eight week undergraduate summer program to provide STEM research opportunities. These students will be trained in consecutive summers and become mentors in their last year. The project will also provide training to pre-and post-doctoral students.The project focuses on the development of an in cell protein footprinting method coupled with mass spectrometry for the characterization of protein interactions inside the cell. A major hindrance in studying protein-lipid interactions is simulating the membrane environment of the cell. The approach this project develops will lead significant advances in studies of membrane proteins by providing a method that can characterize them in their native cellular environment. The method, which uses "caged hydrogen peroxide", will first be developed using a well-characterized cytosolic protein as a model system. Parameters of the method will be systematically optimized and quantitative benchmarks will be used to determine success. Statistical analysis will be used to validate the significance in observed changes in protein modification. Then the membrane protein angiomotin will be used as a model system for the study of transmembrane proteins. This project will result in the development of a novel analytical method for the study of proteins in the cellular environment in living cells. The method will allow for the study of membrane proteins that cannot be studied by currently available structural methods and provide new insight into membrane trafficking in cellular environment.