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。 尽管它们的生物学重要性，膜蛋白的研究已被这些蛋白质的纯化所涉及的困难所阻碍。 由于大多数关于膜蛋白的研究需要这些蛋白处于膜样环境中，因此已经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的创新方法，通过提供一种可以在活细胞中的天然脂质环境中表征膜蛋白的方法，显着推进膜蛋白领域的知识。该方法将首先开发使用一个良好的特性的细胞膜蛋白作为模型系统，然后膜蛋白angiomotin将被用作跨膜蛋白的研究模型系统。该项目将提供一个网关，了解膜贩运的生理学和病理生理学。该项目将在为期八周的本科暑期课程中为历史上黑人学院和大学的学生提供培训，以提供STEM研究机会。 这些学生将在连续的夏天接受培训，并在最后一年成为导师。该项目还将为博士生和博士后学生提供培训，重点是开发一种细胞内蛋白质足迹法，结合质谱法，用于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可以在其天然细胞环境中表征膜蛋白的方法，从而在膜蛋白的研究中取得重大进展。 该方法，它使用“笼过氧化氢”，将首先开发使用一个良好的特点胞质蛋白作为模型系统。该方法的参数将得到系统优化，并将使用定量基准来确定成功与否。将使用统计分析来验证观察到的蛋白质修饰变化的显著性。膜蛋白血管动蛋白将作为研究跨膜蛋白的模型系统。该项目将导致开发一种新的分析方法，用于研究活细胞中细胞环境中的蛋白质。该方法将允许膜蛋白的研究，不能通过目前可用的结构方法进行研究，并提供新的见解膜运输细胞环境。

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.