On methods for probing structure and function of membrane proteins

Doctoral thesis, 2012

The determination of structure–function relationships in membrane proteins plays a key role for the understanding of molecular mechanisms in biological systems, and may provide information on how these systems can be modulated by pharmaceuticals. The aim of the work described herein was to develop methods for studying structure and function of membrane proteins, ideally in combination. The ion channel TRPV1 expressed in Chinese hamster ovary (CHO) cells was used as a model system for evaluation of the methods developed, and further investigations of structure–function relationships in TRPV1 were conducted with these methods. First, a new method for studying ion channel activation was developed based on a microfluidic pipette fabricated in poly(dimethylsiloxane). This device is able to generate a confined volume zone in front of the pipette, and can provide chemical dilution series for the measurement of concentration-response curves in pharmacological studies of cells. The pipette supports delivery of multiple solutions, and rapid switching (<100 ms) between solutions is readily achieved. Second, the pipette was applied in a study were depletion of cholesterol content of cell membranes was found to modulate TRPV1 function under hypocalcemic conditions. The effect of cholesterol-depletion on TRPV1 function was measured with patch-clamp and a fluorescence assay. The treatment was found to inhibit the dynamic permeability of TRPV1 for large cations. Third, a microfluidic flow cell, in which proteoliposomes can be immobilized, was characterized and further developed for use with limited proteolysis. A sequential digestion protocol for proteomic analysis with mass spectrometry of membrane proteins was innovated, where a stationary phase of proteoliposomes derived from CHO-cells was repeatedly exposed to trypsin inside the flow cell channel, and the peptides resulting from each digestion step were used for protein identification. This protocol was found to increase the amount of identified membrane proteins compared to a single-digest protocol, when performing bottom-up proteomic analysis. Finally, a method for the determination of structure–function interconnections in ion channels, combining limited proteolysis with mass spectrometry and patch-clamp recording of ion channel activity, was able to identify distal parts of the TRPV1 protein which are not engaged in activation of the channel with capsaicin.