Intramolecular excitonic dimers in protease substrates: Modification of the backbone moiety to probe the H-dimer structure

NorFES (DAIPN₁SIPKG, N¹ = norleucine) is an undecapeptide that contains a recognition sequence and cleavage site for the serine protease elastase. When NorFES is doubly labeled with a variety of fluorophores on opposite sides of this amino acid sequence, the fluorescence is quenched due to formation of intramolecular ground-state dimers. Although the spectral characteristics of these dimers are predictable by exciton theory. influence of the peptide backbone on H-dimer formation is less well understood. Specifically, factors that modify the attractive forces between and orientation of dyes are not well-characterized. Thus, by varying the dye linker moieties, we have sought to evaluate the thermodynamic parameters for intramolecular H-type dye-dye association and the structures of these dimers. We now present data from a series of homodoubly labeled NorFES derivatives that differ by the addition of one or two 6-aminohexanoic acids to the peptide backbone. By comparing absorption and fluorescence properties of these substrates as a function of temperature, we examined how such additions could modify dimerization: we calculated the free energy of activation (ΔG‡) for intramolecular dimer disruption of each substrate. To gain further insight into dye-dye orientation, a NorFES substrate modified to facilitate intramolecular H-dimerization was synthesized with different geometric dye isomers. The data show that length and conformation of the peptide plus linker as well as stereochemistry of dye-peptide conjugation play important roles in intramolecular ground-state complexation. The factors that influence the spectral properties of intramolecular H-dimerization support our earlier proposed model for H-dimers in NorFES peptides.