SOLVENT ACCESSIBILITY OF THE PHYCOCYANOBILIN CHROMOPHORE IN THE ALPHA-SUBUNIT OF C-PHYCOCYANIN - IMPLICATIONS FOR A MOLECULAR MECHANISM FORINERTIAL PROTEIN-MATRIX SOLVATION DYNAMICS

The solvent environment of the phycocyanobilin chromophore bound by th e a subunit of C-phycocyanin was probed in buffered binary solvent sys tems consisting of water and methanol, acetonitrile, or acetone. The f ocus of the work was on determining whether the inertial phase of the solvent response observed previously in the alpha subunit from femtose cond transient hole-burning spectroscopy [Riter et al. (1996) J. Phys. Chem. 100, 14198-14205] involves solvent dipoles in the bulk. Continu ous absorption and fluorescence spectra at room temperature show that addition of the nonaqueous solvent results in a change in the tertiary structure of the protein so that the phycocyanobilin chromophore is u nclamped and allowed to assume a cyclic conformation. At low concentra tions of nonaqueous solvent, we observe a conformational equilibrium c haracterized by a cooperative binding of nonaqueous solvent. The phyco cyanobilin chromophore exhibits a nonshifted absorption and fluorescen ce spectrum characteristic of its native, extended conformation in the state with bound water molecules. In the state with bound solvent mol ecules, the phycocyanobilin chromophore exhibits an absorption spectru m that reports a cyclic configuration, and its fluorescence is essenti ally quenched. The absorption and fluorescence spectra exhibit a solva tochromic response in this state, indicating that the chromophore is n ow exposed to the bulk solvent. Far-UV circular dichroism spectra evid ence an abrupt loss of 10% of the alpha-helical character in the nonaq ueous solvent concentration regime that results in exposure of the chr omophore to the bulk. These results show that the ultrafast solvation response previously detected in the alpha subunit in aqueous media fro m femtosecond transient hole-burning spectroscopy arises solely from p rotein-matrix solvation dynamics.