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Spontaneous Folding of Human VDAC into Lipid Membranes is Oriented and Indicates a Partially Uncoupled Mechanism

Spontaneous Folding of Human VDAC into Lipid Membranes is Oriented and Indicates a Partially Uncoupled Mechanism

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BALADHANDAPANI, Shanmugavadivu, 2007. Spontaneous Folding of Human VDAC into Lipid Membranes is Oriented and Indicates a Partially Uncoupled Mechanism [Dissertation]. Konstanz: University of Konstanz

@phdthesis{Baladhandapani2007Spont-8665, title={Spontaneous Folding of Human VDAC into Lipid Membranes is Oriented and Indicates a Partially Uncoupled Mechanism}, year={2007}, author={Baladhandapani, Shanmugavadivu}, address={Konstanz}, school={Universität Konstanz} }

2007 terms-of-use 2011-03-24T17:45:32Z eng Baladhandapani, Shanmugavadivu 2011-03-24T17:45:32Z Die spontane Faltung von menschlichem VDAC in Lipidmembranen ist orientiert und beinhaltet einen teilweise unabhängigen Mechanismus application/pdf The entire thesis work presented here concerns the structure, folding and assembly of human mitochondrial outer membrane protein, VDAC and the major outcome of this thesis was summarized in separate sections below.<br />In the first study, initially a protocol for isolation of hVDAC1 in pure form with high yield was established. Next, the hVDAC1 purified in denatured form was successfully refolded to its native conformation and a single-step folding protocol for hVDAC1 was established. During the refolding trials, several important observations were made and the findings are listed below;<br />1. Folding of hVDAC1 into lipid bilayers was spontaneous and did not require any proteinaceous machinery or energy in the form of ATP to assist folding. This would list hVDAC1 as the first eukaryotic -barrel to spontaneously fold into lipid bilayers.<br />2. Also the result implied the earlier finding for soluble proteins that the fold of a protein appears to be solely encoded in the primary amino acid sequence of the protein and this was found to be applicable even to membrane proteins of human origin provided the conditions are not denaturing.<br />3. An interesting and important outcome from this study was that correct-folding of hVDAC1 occurred only in lipid bilayers and not in detergent micelles. Mild detergents like LDAO, DDM and C8E4 commonly used for solubilizing and crystallizing purposes did not allow proper folding of hVDAC1 and this required subsequent reconstitution into membranes.<br />4. hVDAC1 was found to always insert in one direction into the membrane and the finding concluded oriented membrane insertion for hVDAC1.<br />In the next study, the mechanism behind and steps involved in folding and membrane insertion of hVDAC1 were explored. In this study, the kinetics of membrane insertion was analyzed mainly by fluorescence spectroscopy. The results revealed some similarities and several notable differences from OmpA, the existing well established model for prokaryotic β-barrel. The major conclusions are listed below;<br />1. hVDAC1 was largely folded in aqueous buffer and thus further folding and membrane insertion had less restrictive requirements (listed in next points). Further, kinetics of folding and membrane insertion was faster when compared with bacterial β-barrels.<br />2. Most obvious outcome was membrane insertion of hVDAC1 into phosphatidylcholine bilayers like diC12:0PC and diC14:0PC had a low energy barrier and required only low activation energy.<br />3. Membrane insertion of hVDAC1 was found to proceed via two membrane-associated intermediates, first a membrane-adsorbed and second a partially membrane-inserted intermediate.<br />4. Folding and insertion was found to take place independent of the lipid concentration but dependent on lipid bilayer properties like membrane thickness and head group.<br />5. Folding and insertion was found to be sequential and occurred in separate steps. All the above results indicated strong differences in the mechanism and folding pathway for hVDAC1 when compared with bacterial β-barrels. The work also established hVDAC1 as a first model of eukaryotic origin to study the principles of folding and assembly of β-barrel membrane proteins.<br />In the third study, an attempt was made to probe the structure / the transmembrane topology of hVDAC1 in lipid bilayer. The study needed further experiments to analyze the topology. Nevertheless it yielded valuable information regarding the location of a tryptophan residue which is disputed until recently.<br />1. It was found that the tryptophan-209 has a membrane location and inserted into the lipid bilayer. To our knowledge the study was the first experimental proof to confirm the location of tryptophan in hVDAC1 and differed from the existing 13 β-strand model (Colombini 2004) on hVDAC1.<br />2. In addition, the work showed that the intramolecular distance between the -helix and last β-strand in the barrel was at least greater than 15-20 Å. The results would be helpful and be a necessary input for future structural studies on VDAC.<br />In the final study, the effect of lipid bilayer thickness and flexibility was investigated in detail on the example of OmpA. Since membrane thickness was found to largely affect insertion of β-barrel proteins (Kleinschmidt and Tamm 2002a), this study was conducted. The study showed that flexibility, surface curvature and elastic moduli of the lipid affect the activation energy of insertion of proteins. Baladhandapani, Shanmugavadivu Spontaneous Folding of Human VDAC into Lipid Membranes is Oriented and Indicates a Partially Uncoupled Mechanism

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