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.
