Solvent accessibility of the phycocyanobilin chromophore in the alpha subunit of C-phycocyanin: implications for a molecular mechanism for inertial protein-matrix solvation dynamics.

Abstract

The solvent environment of the phycocyanobilin chromophore bound by the alpha subunit of C-phycocyanin was probed in buffered binary solvent systems consisting of water and methanol, acetonitrile, or acetone. The focus of the work was on determining whether the inertial phase of the solvent response observed previously in the alpha subunit from femtosecond transient hole-burning spectroscopy [Riter et al. (1996) J. Phys. Chem. 100, 14198-14205] involves solvent dipoles in the bulk. Continuous 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 unclamped and allowed to assume a cyclic conformation. At low concentrations of nonaqueous solvent, we observe a conformational equilibrium characterized by a cooperative binding of nonaqueous solvent. The phycocyanobilin chromophore exhibits a nonshifted absorption and fluorescence spectrum characteristic of its native, extended conformation in the state with bound water molecules. In the state with bound solvent molecules, the phycocyanobilin chromophore exhibits an absorption spectrum that reports a cyclic configuration, and its fluorescence is essentially quenched. The absorption and fluorescence spectra exhibit a solvatochromic response in this state, indicating that the chromophore is now exposed to the bulk solvent. Far-UV circular dichroism spectra evidence an abrupt loss of 10% of the alpha-helical character in the nonaqueous solvent concentration regime that results in exposure of the chromophore to the bulk. These results show that the ultrafast solvation response previously detected in the alpha subunit in aqueous media from femtosecond transient hole-burning spectroscopy arises solely from protein-matrix solvation dynamics.