INTRAMOLECULAR EXCITONIC DIMERS IN PROTEASE SUBSTRATES - MODIFICATIONOF THE BACKBONE MOIETY TO PROBE THE H-DIMER STRUCTURE

NorFES (DAIPN(1)SIPKGY, N-1 norleucine) is an undecapeptide that conta ins a recognition sequence and cleavage site for the serine protease e lastase. When NorFES is doubly labeled with a variety of fluorophores on opposite sides of this amino acid sequence, the fluorescence is que nched due to formation of intramolecular ground-state dimers. Although the spectral characteristics of these dimers are predictable by excit on theory, influence of the peptide backbone on H-dimer formation is l ess well understood, Specifically, factors that modify the attractive forces between and orientation of dyes are not well-characterized. Thu s, 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 compar ing absorption and fluorescence properties of these substrates as a fu nction of temperature, we examined how such additions could modify dim erization; we calculated the free energy of activation (Delta G(double dagger)) for intramolecular dimer disruption of each substrate, To ga in further insight into dye-dye orientation, a NorFES substrate modifi ed to facilitate intramolecular H-dimerization was synthesized with di fferent geometric dye isomers. The data show that length and conformat ion of the peptide plus linker as well as stereochemistry of dye-pepti de conjugation play important roles in intramolecular ground-state com plexation. The factors that influence the spectral properties of intra molecular H-dimerization support our earlier proposed model for H-dime rs in NorFES peptides.