Uncovering dehydration in cytochrome <i>c</i> refolding from urea- and guanidine hydrochloride-denatured unfolded state by high pressure spectroscopy

Abstract

To investigate the dehydration associated with protein folding, the partial molar volume changes for protein unfolding (ΔV u ) in cytochrome c (Cyt c) were determined using high pressure absorption spectroscopy. ΔV u values for the unfolding to urea-and guanidine hydrochloride (GdnHCl)-denatured Cyt c were estimated to be 56±5 and 29±1 mL mol -1 , respectively. Considering that the volume change for hydration of hydrophobic groups is positive and that Cyt c has a covalently bonded heme, a positive ΔV u 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 c than in urea-denatured Cyt c, resulting in the smaller positive ΔV u . On the other hand, urea is a relatively weak denaturant and urea-denatured Cyt c 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 ΔV u (4±2 mL mol -1 ) in urea-denatured Cyt c. 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 c by estimating ΔV u , which allows us to discuss the hydrated structures in the denatured states of proteins.In aqueous solutions, linear protein polypeptide chains decrease in entropy and collapse into a globule to minimize the surface area that is exposed to the solvent. The folded state is a low-entropy subensemble in all possible collapsed globular conformations for the protein chain. In contrast, the unfolded state is an ensemble that is not or much less structured and has higher entropy than the folded state does. For folding to be beneficial, the folded state must be sufficiently and energetically favorable to overwhelm the higher entropy associated with structural disorder within the globular phase. The major energetic driving force originates from the van der Waals, hydrogen bonding, and electrostatic interactions, both within the polypeptide chain and between the chain andThe high pressure absorption spectroscopy revealed that the partial volume changes for the unfolding of cytochrome c (Cyt c) to the urea-and guanidine hydrochloride (GdnHCl)-denatured unfolded states (ΔV u ) were positive, reflecting hydration to hydrophobic heme; however, a more positively shifted ΔV u was observed for urea-denatured Cyt c. Introduction of the mutation near the axial ligand induced more drastic changes in the hydrated structure of the urea-denatured Cyt c, suggesting that the hydrated structure in the unfolded state depends on the denaturant. Our approach enables us to examine the dehydration associated with protein folding and hydration structures in the unfolded states.

Activation of Cytochrome C Peroxidase Function Through Coordinated Foldon Loop Dynamics upon Interaction with Anionic Lipids

“…The impact of membranes on cyt c has often been discussed as a process of (partial) unfolding [28,47,52,61], with potential analogy to denaturation of cyt c in solution [59,65,66]. In apparent support of this conclusion, an effective CL/cyt c ratio of 25 (L/P=100) causes a similar fluorescence emission to that of GdHCl denatured cyt c ( Figure S1c).…”

“…In vitro, there are many studies reporting on the interactions of cyt c with non-CL anionic lipids, including PG, showing the potential for similar interactions to those underpinning CL-induced peroxidase activation [28][29][30][31]. Yet, to the best of our knowledge there is no record of a direct involvement of PG in place of CL in the apoptotic pathway in vivo, with studies showing CL to be the preferred substrate for cyt c binding and peroxidation [2,3,32,33] [34]: the blue foldon (N and C-terminal helices, residues 1-14 and 88-104), the green foldon (helix [61][62][63][64][65][66][67][68][69] and Ω loop [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35], the gray foldon (Ω loop [40][41][42][43][44][45][46][47][48][49][50][51]…”

Biophysical research in Hokkaido University, Japan

“…Koichi Ishimori, Takeshi Uchida, and Tomohide Saio. They work to functionally and structurally characterize key proteins essential for biological systems including electron transfer, metal homeostasis, protein folding, and various kinds of chemical processes in cells (Uchida et al 2018;Konno et al 2019). They have also been developing new spectroscopic methodologies to unveil the dynamic structural and functional properties of proteins using NMR, resonance Raman, CD, and time-resolved techniques (Saio and Ishimori 2019).…”