HDX of Membrane Proteins

Project Abstract

Aquaporin-0 (AQP0) is the major integral membrane protein of ocular lens fiber cells. It is a water channel as well as a structural protein with cell-cell adhesion properties. Since there is no protein turnover in lens fiber cells, old proteins in the inner nucleus are heavily modified by posttranslational modification whereas proteins in the outer cortex are newly synthesized and lightly modified. AQP0 water permeability is regulated by calmodulin and specific sites of interaction between the AQP0 C-terminal tail and calmodulin have been determined in the Schey lab by chemical crosslinking-mass spectrometry experiments. The goals of this project are to: 1) develop methodology for HDX experiments on AQP0 that will be generally applicable to other integral membrane proteins, 2) measure age-related structural changes by comparing HDX rates in regions of AQP0 purified from outer cortical cells and inner nuclear cells, and 3) determine additional sites of interaction with calmodulin and putative conformation changes. Studies will be carried out using AQP0 purified from dissected bovine lenses. Samples will be prepared using the fully automated HDX system from Waters and data will be acquired on a Waters Synapt G2si Q-TOF mass spectrometer. Data analysis will be accomplished with Waters DynamX software.

Training Plan

Dr. Schey will meet twice weekly with the trainee; once in lab meeting and once with the small group of HDX researchers. Dr. Schey will supervise all aspects of the project, assist with experimental design, and assist with data interpretation. Dr. Zhen Wang in the Schey lab will assist with AQP0 purification from bovine lenses and Ms. Wendy White in the Proteomics Core will assist with data acquisition and data analysis. The trainee will be expected to discuss his results on a weekly basis at lab meetings. He will be expected to read the literature on AQP0 and HDX analysis.

Mentors

Primary: Kevin Schey