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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Some <strong>of</strong> the biological questions debated here are still open. Recently, a model for<br />
the binding site <strong>of</strong> V-ATPase inhibitors in the c-subunit was proposed (Bowman et al.,<br />
2006), confirming that residues in at least three different trans<strong>membrane</strong> domains were<br />
required for effective bafilomycin inhibition. However the picture was not completely<br />
clear, as residues in opposite sides <strong>of</strong> the same helix were found to be relevant for<br />
bafilomycin binding pointing to indirect effects <strong>of</strong> some residues in the interaction <strong>with</strong><br />
the inhibitor. Although, clinical trials <strong>with</strong> SB242784 were not started due to possible<br />
adverse effects (Nikura et al., 2005), a derivative <strong>of</strong> this molecule was shown recently to<br />
potentiate the effect <strong>of</strong> an cytotoxic/anti-tumor drug likely due to inhibition <strong>of</strong> V-<br />
ATPases in tumor cells (Petrangoline et al., 2006).<br />
The study <strong>of</strong> the mechanisms behind clustering <strong>of</strong> PI(4,5)P 2 into cholesterol<br />
dependent domains in bio<strong>membrane</strong>s is a very promising field. It is clear that PI(4,5)P 2<br />
should not spontaneously cluster or partitionate to liquid ordered domains, and this<br />
behaviour must be connected to specific protein-lipid interactions. In this way, the study<br />
<strong>of</strong> the lipid <strong>membrane</strong> partition properties <strong>of</strong> <strong>proteins</strong> known to bind PI(4,5)P 2 might<br />
give us some insight into these mechanisms. The relevance <strong>of</strong> acylation characteristics<br />
(number and length <strong>of</strong> acyl-chains in the protein), for <strong>membrane</strong> protein organization in<br />
bio<strong>membrane</strong>s, is a problem <strong>of</strong> great biological relevance.<br />
The use <strong>of</strong> fluorescence imaging techniques in the study <strong>of</strong> protein-lipid interactions<br />
can add additional layers <strong>of</strong> information to the results <strong>of</strong> non-space resolution<br />
biophysical <strong>studies</strong>. The biophysical <strong>studies</strong> presented here relied mostly on<br />
macroscopic observables, and although this type <strong>of</strong> data is, as demonstrated, highly<br />
enriched in information, some <strong>of</strong> it is lost by averaging <strong>of</strong> fluorophore populations,<br />
while imaging techniques allow for resolution <strong>of</strong> different populations <strong>of</strong> fluorophores.<br />
Therefore, the application <strong>of</strong> the methodologies derived and described here to<br />
fluorescence imaging data could help identify features <strong>of</strong> interactions between<br />
<strong>membrane</strong> components which go undetected in the macroscopic data. In the limit,<br />
single-molecule techniques could also be used. Imaging techniques are obviously very<br />
useful in the observation <strong>of</strong> large scale changes (µm scale) in the lateral distribution <strong>of</strong><br />
<strong>membrane</strong> components and are a useful complement to FRET methodologies, which are<br />
able to probe deviations to homogeneity in the nanometer scale. Additionally, FRET<br />
<strong>studies</strong> under the microscope are also carried out, allowing the direct comparison<br />
between macroscopic and microscopic data.<br />
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