Small, flexible peptides mediate a broad spectrum of biological processes in human tissues from brain to gut. Even a single peptide can evoke different responses depending on the target tissue. Of the many possible conformations in solution, only a few will be biologically active conformers recognized by specific receptors. Structural determination of flexible peptides is thus an important challenge for structure-based drug design. This proposal develops fluorescence techniques for studying the structure and dynamics of flexible peptides in solution and in membrane receptor complexes. Our approach features the design of novel fluorescence probes that mimic the naturally occurring aromatic amino acids while minimally perturbing peptide structure and activity. These probes in turn are the gateway to understanding the relationship between fluorescence lifetime and ground-state structure. Excited-state properties are determined by time-correlated single photon counting. Ground-state structure is determined by X-ray crystallography, NMR, and molecular mechanics. Fundamental studies of tryptophan photophysics will dissect the complex photophysics of the indole chromophore. Nonradiative processes responsible for the environmental sensitivity of the fluorescence lifetime include: solvent quenching and excited-state proton and electron transfer reactions. Strategies for identifying, separating, and quantifying several competitive nonradiative processes will be used to map all possible quenching interactions between the indole ring and amino acid functional groups. Fluorescence quenching mechanisms will be delineated by solvent isotope effect, temperature dependence, and photochemical isotope exchange experiments. Second generation constrained tryptophan derivatives that mimic all significant tryptophan conformations in peptides will be synthesized. These probes will define the proximity and orientation requirements and mechanism of tryptophan fluorescence quenching by peptide bonds. Distance and orientation requirements for intramolecular quenching of tryptophan fluorescence by amino acid functional groups will be systematically studied in real peptides. Constrained and benzannulated tryptophan derivatives and amino acids whose side chains quench indole fluorescence will be incorporated into rigid somatostatin analogs. Quenching mechanisms will be characterized. Excited-state lifetimes will be related to solution conformations determined by high resolution NMR and free energy molecular mechanics. A benzannulated tryptophan derivative will be developed to probe peptide structure in complex environments, such as membrane receptors. Amino acids with acrylamide-like functional groups will be incorporated as intramolecular quenchers of benzannulated indole fluorescence. This work tests the methodology for peptide structure determination in peptides of known structure. Future work will involve application to flexible peptides in solution and bound to membrane receptors.
