Bz. Packard et al., INTRAMOLECULAR EXCITONIC DIMERS IN PROTEASE SUBSTRATES - MODIFICATIONOF THE BACKBONE MOIETY TO PROBE THE H-DIMER STRUCTURE, JOURNAL OF PHYSICAL CHEMISTRY B, 102(10), 1998, pp. 1820-1827
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.