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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FINAL CONSIDERATIONS AND PROSPECTS<br />
VIII<br />
FINAL CONSIDERATIONS AND<br />
PROSPECTS<br />
<strong>Biophysical</strong> <strong>studies</strong> are likely the only possibility <strong>of</strong> direct structural and dynamic<br />
characterization <strong>of</strong> the type <strong>of</strong> interactions addressed in this work. The recurrent<br />
difficulties in <strong>membrane</strong> protein expression and purification were responsible for the<br />
popularity <strong>of</strong> the application <strong>of</strong> highly sensitive fluorescence techniques in <strong>studies</strong> <strong>of</strong><br />
<strong>membrane</strong> <strong>proteins</strong>. The presence <strong>of</strong> aromatic amino acids in <strong>proteins</strong> allows the<br />
application <strong>of</strong> fluorescence techniques <strong>with</strong>out requirement for labelling <strong>with</strong> a extrinsic<br />
fluorophore. Still, when necessary, the developments in synthesis <strong>of</strong> fluorophores and<br />
labelling techniques, provide the researcher <strong>with</strong> great flexibility in the process <strong>of</strong><br />
choosing the best conditions for fluorescence methodologies. Overall, the <strong>studies</strong><br />
presented here are a demonstration <strong>of</strong> the potential <strong>of</strong> biophysical <strong>studies</strong>, particularly<br />
fluorescence spectroscopy <strong>studies</strong>, in the characterization <strong>of</strong> interactions between<br />
components <strong>of</strong> bio<strong>membrane</strong>s.<br />
In this work, the use <strong>of</strong> <strong>membrane</strong> model systems, allowed for the simplification <strong>of</strong><br />
the number <strong>of</strong> variables in the experiments. This is essential in <strong>studies</strong> that focus on the<br />
characterization <strong>of</strong> very specific interactions. Apart from the technical difficulties<br />
inherent to in vivo <strong>studies</strong> (huge heterogeneity <strong>of</strong> samples, background fluorescence,<br />
light scattering, low fluorescence intensity, etc.), the presence <strong>of</strong> a large number <strong>of</strong><br />
uncontrolled (and sometimes unknown) variables is highly likely to hinder the<br />
possibility <strong>of</strong> data rationalization. The information recovered from <strong>studies</strong> <strong>with</strong> less<br />
complex model systems can then be used for a more clear rationalization <strong>of</strong> data<br />
gathered from in vivo <strong>studies</strong>.<br />
The new FRET methodologies developed here, notably the methodology for<br />
quantification <strong>of</strong> protein-lipid selectivity, are expected to be <strong>of</strong> great use in the future as<br />
they are readily applicable to different systems. The FRET methodologies for<br />
quantification <strong>of</strong> drug-protein binding will require only some straightforward<br />
adaptations to the geometric constraints <strong>of</strong> the systems to be studied.<br />
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