Research objectives and content
The aim of the research described in this proposal is to determine the influence of lipid lateral forces in membranes on the folding, stability and enzymatic activity of membrane proteins. As a model system we will use bacteriorhodopsin in combination with lipid bilayers consisting of mixtures of the C6-chain lipid dihexanoylphosphatidylcholine (DHPC) and the C14-chain lipid dimyristoylphosphatidylcholine. In this system the lateral pressure exerted by the lipid molecules on the protein can be can be controlled by varying the molar ratio of the two components. We will determine
a) the physical properties of the bilayer in varying compositions and in the presence and absence of the protein and b) the folding properties, stability and activity of the protein as a function of changes in the bilayer composition.
The physical properties of the bilayer (a) will be determined by differential scanning calorimetry, isothermal titration calorimetry, excimer fluorescence of inserted di-pyrene probes and X-ray diffraction. The folding properties and activity of the protein (b) will be determined spectroscopically (fluorescence, absorption and circular dichroism). The stability of the protein (b) will be evaluated using differential scanning calorimetry.
Training content (objective, benefit and expected impact)
There is good complementarity between the skills of the applicant and the training provided by the host institution, and therefore both will benefit from their cooperation. The applicant brings experience in the use of calorimetric techniques in combination with membrane proteins as well as a an understanding of the thermodynamics of protein stability. The host institute will provide training in the use of time-resolved techniques in the study of enzymatic activity and protein folding and the analysis of properties of lipids in membranes. Understanding of the interactions between lipids and membrane proteins can give us a tool to control enzyme activity in the membrane, or to understand how this control is achieved in living cells. It has also been shown that in the crystallisation process of membrane proteins lipid forces play an important role and therefore a better understanding of these intermolecular interactions can facilitate the structural analysis of this class of proteins.
Links with industry / industrial relevance (22)