Methods for elucidating membrane protein structure and function
Doktorsavhandling, 2015

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.

patch clamp

antibodies

mass spectrometry

ion channels

proteomics

fluorescence

TRPV1

cholesterol.

limited proteolysis

Membrane proteins

KB
Opponent: Prof. Jonas Bergquist

Författare

Carolina Trkulja

Chalmers, Kemi och kemiteknik, Kemi och biokemi, Fysikalisk kemi

Probing Structure and Function of Ion Channels Using Limited Proteolysis and Microfluidics

Journal of the American Chemical Society,; Vol. 136(2014)p. 14875-14882

Artikel i vetenskaplig tidskrift

Effect of cholesterol depletion on the pore dilation of TRPV1

Molecular Pain,; Vol. 9(2013)

Artikel i vetenskaplig tidskrift

Microfluidic Flow Cell for Sequential Digestion of Immobilized Proteoliposomes

Analytical Chemistry,; Vol. 84(2012)p. 5582-5588

Artikel i vetenskaplig tidskrift

Ämneskategorier

Fysikalisk kemi

Cellbiologi

Biokemi och molekylärbiologi

Styrkeområden

Livsvetenskaper och teknik

ISBN

978-91-7597-247-3

Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie: 3928

KB

Opponent: Prof. Jonas Bergquist

Mer information

Skapat

2017-10-07