HDX-MS  Studies of Integral Membrane Proteins

Project Abstract

The goal of this project is to develop methods for HDX-MS experiments on integral membrane proteins. An abundant, accessible integral membrane protein, ocular lens aquaporin-0, will be used as the model system to optimize sample preparation conditions, digestion conditions, and data acquisition methods for HDX-MS. Bovine lens AQP0 will be purified by anion exchange chromatography and subjected to HDX-MS as a function of exchange time to determine regions of highest exposure and whether membrane spanning regions undergo exchange. With optimized HDX-MS methods, studies of AQP0 structural perturbations and protein-protein interactions will be examined. Of high interest is the effect of cholesterol on AQP0 structure and the interaction of AQP0 with calmodulin.

Training Plan

Ary Furman will work closely with Marcus Nagel in the Schey lab to learn the HDX-MS protocol. He will also work with members of the Schey to purify bovine lens AQP0; a routine procedure in the Schey lab. He will attend and present progress reports at the weekly Schey lab meetings and will attend monthly joint Schey/Meiler/Crowe lab meetings to learn about the role of HDX-MS in structural biology. Ary will also work with Marcus Nagel to learn protein structure visualization tools and membrane protein modeling tools in order to place his HDX-MS work in a structural biology context. He will present his work at the VSURP poster session at the end of the summer. Dr. Schey will supervise all HDX-MS experiments. Dr. Meiler will advise on structural biology and membrane protein modeling aspects of the project.

Mentors

Primary: Kevin Schey

Secondary: Jens Meiler