A study of the spontaneous membrane insertion of chloride intracellular ion channel protein CLIC1 into model lipid membranes
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Sterols have been reported to modulate conformation and hence the function of several membrane proteins. One such group is the Chloride Intracellular Ion Channel (CLIC) family of proteins. These largely soluble proteins possess the intriguing property of spontaneous insertion into phospholipid bilayers to form integral membrane ion channels. To date, the structure of their membrane-bound form and factors influencing their auto-insertion remains largely unknown. In this thesis, we have performed Langmuir-film, X-ray, and neutron reflectivity experiments to study the interaction of wild-type or mutant versions of the protein CLIC1 with monolayers prepared using various mixtures of different phospholipids and sterol molecules, in order to investigate the regulatory role of the membrane lipid combination on the spontaneous membrane insertion of CLIC1 and to elucidate the structural features of the CLIC1 membrane-bound form within the lipid monolayers. Our findings have demonstrated that the spontaneous membrane insertion of CLIC1 is dependent on the presence of cholesterol in lipid monolayers. In phospholipid monolayers only, CLIC1 was able to insert within the phospholipid head-group region with no penetration into the acyl chain region of the monolayers. However, in the presence of cholesterol, CLIC1 showed significant interaction with the phospholipid acyl chains thereby, suggesting that cholesterol is required for the penetration of CLIC1 into the hydrophobic tails of the lipid monolayer, which is considered necessary for the formation of functional ion channels. From reflectivity experiments, we were able to show that approximately 0.8 mg/m² of CLIC1 inserted into phospholipid monolayers containing cholesterol such that the protein occupied an area per molecule between 5 ~ 7 nm² with a total CLIC1 thickness ranging from ~ 51 Å to 59 Å throughout the entire monolayer. We have also demonstrated for the first time that the GXXXG motif in CLIC1 acts as the cholesterol-binding site used by the protein for its initial recognition and binding to membrane cholesterol. Furthermore, Langmuir and reflectivity experiments using different sterols have confirmed that the interaction between CLIC1 and sterols is dependent on an intact 3β-OH group in the sterol ring. Modification of the sterol structure by the introduction of additional hydroxyl groups and methylation of the sterol alkyl chain was shown to facilitate greater spontaneous membrane insertion of the protein within the phospholipid monolayer. Taken together these findings provide clear evidence for the important role of sterols in the regulation of CLIC1 membrane interactions and a putative mechanism for its initial binding and membrane integration.
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