Biophysical studies of membrane proteins/peptides. Interaction with ...
Biophysical studies of membrane proteins/peptides. Interaction with ...
Biophysical studies of membrane proteins/peptides. Interaction with ...
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OUTLINE<br />
<strong>membrane</strong>s <strong>with</strong> a distribution <strong>of</strong> donors and acceptors, FRET will be sensitive to<br />
changes in the distribution <strong>of</strong> acceptors around the donor population in the space range<br />
<strong>of</strong> the Förster radius (up to ~10 nm depending on the donor-acceptor pair).<br />
Several different systems were investigated. In Chapter II, an integral protein from<br />
the coat <strong>of</strong> the bacteriophage M13 (mcp) was studied in model <strong>membrane</strong>s. The<br />
oligomerization state <strong>of</strong> the protein changes repeatedly during the reproductive cycle <strong>of</strong><br />
bacteriophage M13, but the state <strong>of</strong> the protein in the <strong>membrane</strong> was thought to be<br />
monomeric as this is apparently required for correct insertion in nascent bacteriophages.<br />
However, this was still matter <strong>of</strong> debate, as in the past, oligomeric mcp was shown to<br />
exist in some conditions. Here, it is shown that mcp is a monomer when incorporated in<br />
<strong>membrane</strong>s composed by lipids <strong>with</strong> hydrophobic thickness that matched the<br />
hydrophobic thickness <strong>of</strong> the protein (monounsatured chains C 18 chains). On the other<br />
hand, when bilayers <strong>with</strong> thicker hydrophobic thickness (monounsatured chains C 22<br />
chains) were used, aggregation was observed. Surprisingly, when mixtures <strong>of</strong> both<br />
matching and mismatching lipids were used, segregation <strong>of</strong> mcp to domains enriched in<br />
matching lipid was apparently induced, and while in these domains, mcp remained<br />
monomeric. This result is a dramatic demonstration <strong>of</strong> the relevance <strong>of</strong> hydrophobic<br />
matching for protein-lipid organization. Only a small increase <strong>of</strong> 4 carbons in the acylchain<br />
<strong>of</strong> the matrix lipid can drive protein aggregation, and in case that a significant<br />
fraction <strong>of</strong> matching lipid is present, lipid-lipid segregation can be induced by the<br />
presence <strong>of</strong> the protein.<br />
A FRET methodology was developed to quantify the degree <strong>of</strong> selectivity exhibited<br />
by mcp to different lipids. The results obtained for lipids <strong>with</strong> different headgroups are<br />
almost perfectly matched to previous results obtained <strong>with</strong> electron spin resonance<br />
(ESR) for the same protein, even though in an aggregated state. This agreement<br />
establishes the FRET methodology for quantification <strong>of</strong> protein-lipid selectivity<br />
described here as an alternative to the use <strong>of</strong> ESR. This FRET methodology was also<br />
applied to the study <strong>of</strong> selectivity for acyl-chain length, which is as already mentioned,<br />
essential in dictating the lateral distribution <strong>of</strong> the protein in liposomes. Also<br />
surprisingly, the results revealed weak selectivity constants for a hydrophobically<br />
matching lipid, both in thinner as in thicker bilayers.<br />
Drug binding to <strong>membrane</strong> <strong>proteins</strong> was studied following two different strategies.<br />
In chapter III, a reductionist approach was used. A peptide expected to correspond to the<br />
putative 4 th trans<strong>membrane</strong> domain <strong>of</strong> the <strong>membrane</strong> bound c-subunit <strong>of</strong> V-ATPase,<br />
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