Konno Shohei, Doi Kentaro, Ishimori Koichiro
Graduate School of Chemical Sciences and Engineering, Hokkaido University, Sapporo, Hokkaido 060-8628, Japan.
Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo, Hokkaido 060-0810, Japan.
Biophys Physicobiol. 2019 Jan 31;16:18-27. doi: 10.2142/biophysico.16.0_18. eCollection 2019.
To investigate the dehydration associated with protein folding, the partial molar volume changes for protein unfolding (Δ ) in cytochrome (Cyt ) were determined using high pressure absorption spectroscopy. Δ values for the unfolding to urea- and guanidine hydrochloride (GdnHCl)-denatured Cyt were estimated to be 56±5 and 29±1 mL mol, respectively. Considering that the volume change for hydration of hydrophobic groups is positive and that Cyt has a covalently bonded heme, a positive Δ reflects the primary contribution of the hydration of heme. Because of the marked tendency of guanidium ions to interact with hydrophobic groups, a smaller number of water molecules were hydrated with hydrophobic groups in GdnHCl-denatured Cyt than in urea-denatured Cyt , resulting in the smaller positive Δ . On the other hand, urea is a relatively weak denaturant and urea-denatured Cyt is not completely hydrated, which retains the partially folded structures. To unfold such partial structures, we introduced a mutation near the heme binding site, His26, to Gln, resulting in a negatively shifted Δ (4±2 mL mol) in urea-denatured Cyt . The formation of the more solvated and less structured state in the urea-denatured mutant enhanced hydration to the hydrophilic groups in the unfolding process. Therefore, we confirmed the hydration of amino acid residues in the protein unfolding of Cyt by estimating Δ , which allows us to discuss the hydrated structures in the denatured states of proteins.
为了研究与蛋白质折叠相关的脱水过程，我们使用高压吸收光谱法测定了细胞色素c（Cyt c）蛋白质解折叠的偏摩尔体积变化（ΔV）。Cyt c解折叠为尿素和盐酸胍（GdnHCl）变性态时的ΔV值分别估计为56±5和29±1 mL/mol。考虑到疏水基团水合的体积变化为正值，且Cyt c含有共价结合的血红素，正的ΔV反映了血红素水合的主要贡献。由于胍离子与疏水基团相互作用的显著倾向，与尿素变性的Cyt c相比，GdnHCl变性的Cyt c中与疏水基团水合的水分子数量更少，导致正的ΔV更小。另一方面，尿素是一种相对较弱的变性剂，尿素变性的Cyt c没有完全水合，保留了部分折叠结构。为了展开这种部分结构，我们将血红素结合位点附近的His26突变为Gln，导致尿素变性的Cyt c中ΔV向负方向移动（4±2 mL/mol）。尿素变性突变体中形成的更易溶剂化且结构更少的状态在解折叠过程中增强了对亲水基团的水合作用。因此，我们通过估计ΔV证实了Cyt c蛋白质解折叠过程中氨基酸残基的水合作用，这使我们能够讨论蛋白质变性状态下的水合结构。
