Methods for elucidating membrane protein structure and function

Abstract: Elucidation of the structure-function relationship of membrane proteins increases our understanding of many biological processes, both physiological and pathological. However, membrane proteins have been notoriously difficult to study, having part of their structure embedded into the lipid cell membrane. The work presented herein describes related functional and structural studies of the human ion channel TRPV1 as well as method development for studies on membrane proteins. First, the effect of membrane cholesterol levels on TRPV1 function was evaluated with patch-clamp recordings and cholesterol-depletion inhibited a time-dependent increase in permeability of large cations. Secondly, a microfluidic flow cell was developed for proteomic studies of membrane proteins where proteoliposomes derived from cell membranes were immobilized and constituted a stationary phase of membrane proteins. In addition, a sequential digestion protocol for limited proteolysis within the flow cell was developed for proteomic profiling and digested peptides were identified with mass spectrometry. Moreover, limited proteolysis was performed on TRPV1 for evaluation of surface exposed regions. Identical digestions were performed during inside-out patch-clamp experiments in order to correlate TRPV1 structure with function. Removal of intracellular peptide regions decreased ion channel activity. Finally, the flow cell was employed for generation of antibody epitopes suitable for development of therapeutic antibodies. Two antibodies were raised against intracellular domains of TRPV1 and were further evaluated for their effect on TRPV1 activity using patch-clamp and fluorescence studies. Both antibodies altered TRPV1 activity. This methodology may thus constitute an effective strategy for generation of therapeutic antibodies.