Steady-state and time-resolved fluorescence spectroscopy were employed in the study of the structure and interactions of α-MSH (α-melanocyte-stimulating hormone) and its analogs, [Nle4, d-Phe7]-α-MSH (MSH-I) and [formula omitted] (MSH-II). In aqueous buffer, the fluorescence parameters of the single tryptophan of α-MSH and MSH-I were similar and did not allow any distinction between these molecules. On the other hand, the tryptophan fluorescence of MSH-II was notably different, reflecting its cyclic lactam turn structure. In the presence of acidic lipid vesicles, the fluorescence properties of the peptides were different, indicating structural changes on incorporation of the peptide into the liquid-crystalline phase of the lipid. No evidence of interaction was observed in the presence of the neutral lipid dimyristoylphosphatidylcholine (DMPC). The association constants for lipid-peptide interactions were compared for binding isotherms which either neglected or accounted for electrostatic effects through Gouy-Chapman potential functions. The relative order of association constants in either treatment was MSH-II > MSH-I > α-MSH. These results parallel the reported biological activities that show increased potencies and prolongation of response for the analogs, MSH-II and MSH-I, as compared to the native hormone, α-MSH. Time-resolved fluorescence results showed that the fluorescence decay of melanotropins is best described by triple-exponential kinetics. In the lipid-peptide complex, there was a change in the relative concentrations of the components, with the intermediate-lifetime component predominating compared to those in solution. The results are consistent with a model where the peptides are initially attracted electrostatically to the vesicle surface and are then incorporated into the lipid phase, due to hydrophobic forces with accompanying conformational changes, to form a compact reversed turn structure.
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