Homoelle B J, Beck W F
Department of Chemistry, Vanderbilt University, 5134 Stevenson Center, Post Office Box 1822-B, Nashville, Tennessee 37235, USA.
Biochemistry. 1997 Oct 21;36(42):12970-5. doi: 10.1021/bi971121n.
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.
在由水与甲醇、乙腈或丙酮组成的缓冲二元溶剂体系中，对与C-藻蓝蛋白α亚基结合的藻胆青素发色团的溶剂环境进行了探测。这项工作的重点是确定先前通过飞秒瞬态空穴烧蚀光谱法在α亚基中观察到的溶剂响应的惯性阶段是否涉及本体中的溶剂偶极子。室温下的连续吸收光谱和荧光光谱表明，添加非水溶剂会导致蛋白质三级结构发生变化，从而使藻胆青素发色团松开并呈现环状构象。在低浓度非水溶剂中，我们观察到一种以非水溶剂协同结合为特征的构象平衡。藻胆青素发色团在与水分子结合的状态下呈现出其天然伸展构象的无位移吸收光谱和荧光光谱特征。在与溶剂分子结合的状态下，藻胆青素发色团呈现出反映环状结构的吸收光谱，并且其荧光基本淬灭。在此状态下，吸收光谱和荧光光谱呈现出溶剂化显色响应，表明发色团现在暴露于本体溶剂中。远紫外圆二色光谱证明，在非水溶剂浓度范围内，α-螺旋特征突然损失10%，导致发色团暴露于本体中。这些结果表明，先前通过飞秒瞬态空穴烧蚀光谱法在水性介质中的α亚基中检测到的超快溶剂化响应仅源于蛋白质-基质的溶剂化动力学。
