Influence Of Solvent Conditions Research Articles

Conformational Heterogeneity of Cytochrome c Probed by Resonance Raman Spectroscopy as a function of pH and Temperature

The oxidized state of cytochrome c is a subject of continuous interest due to the multitude of conformations the protein adopts. Despite numerous studies, native and non-native states of ferricytochrome c have not been comprehensively analyzed regarding the influence of solvent conditions on structure, function, and thermodynamic equilibrium. Compared to the oxidized state, the reduced state of cytochrome c is rather stable since it adopts one conformation over a broad pH (2-12) and temperature (∼100°C) range. In the current study, we have analyzed the high frequency (1200-1800cm−1) Soret and Q-band resonance Raman spectra of oxidized and reduced horse heart cytochrome c (hhc) in terms of depolarization ratios and normalized Raman intensities, as a function of increasing pH and temperature. Initial analysis of our data collected for the Soret band resonance indicates that the depolarization ratios of A1g modes v2, v3, and v4 of the native state III and the alkaline state IV are practically identical. They deviate from the respective D4h-value, indicating that B1g (triclinic) or B2g (rhombic) type distortions affect the Raman tensor. For state III, the depolarization ratios of B1g modes v10, v11, and v13 deviate substantially from the expectation value indicating that these modes are affected by a large B1g type distortion. The alkaline III-> IV transition moves the depolarization ratios of these B1g modes closer to their D4h-value of 0.75, indicating a substantial decrease of the rhombic B1g-type deformation. Data from pH 12, which favor the population of the V-state, suggest a substantial increase of rhombic deformations, apparently caused by the replacement of a lysine by a hydroxyl ligand. Currently we are analyzing the depolarization ratios for the Q band resonance and the high temperature data for both resonances.

The Conformational Manifold of Ferricytochrome c Explored by Visible and Far-UV Electronic Circular Dichroism Spectroscopy

The oxidized state of cytochrome c is a subject of continuous interest, owing to the multitude of conformations which the protein can adopt in solution and on surfaces of artificial and cell membranes. The structural diversity corresponds to a variety of functions in electron transfer, peroxidase and apoptosis processes. In spite of numerous studies, a comprehensive analysis and comparison of native and non-native states of ferricytochrome c has thus far not been achieved. This results in part from the fact that the influence of solvent conditions (i.e., ionic strength, anion concentration, temperature dependence of pH values) on structure, function and equilibrium thermodynamics has not yet been thoroughly assessed. The current study is a first step in this direction, in that it provides the necessary experimental data to compare different non-native states adopted at high temperature and alkaline pH. To this end, we employed visible electronic circular dichroism (ECD) and absorption spectroscopy to probe structural changes of the heme environment in bovine and horse heart ferricytochrome c as a function of temperature between 278 and 363 K at different neutral and alkaline pH values. A careful selection of buffers enabled us to monitor the partial unfolding of the native state at room temperature while avoiding a change to an alkaline state at high temperatures. We found compelling evidence for the existence of a thermodynamic intermediate of the thermal unfolding/folding process, termed III h, which is structurally different from the alkaline states, IV 1 and IV 2, contrary to current belief. At neutral or slightly acidic pH, III h is populated in a temperature region between 320 and 345 K. The unfolded state of the protein becomes populated at higher temperatures. The ECD spectra of the B-bands of bovine and horse heart cytochrome c (pH 7.0) exhibit a pronounced couplet that is maintained below 343 K, before protein unfolding replaces it by a rather strong positive Cotton band. A preliminary vibronic analysis of the B-band profile reveals that the couplet reflects a B-band splitting of 350 cm (-1), which is mostly of electronic origin, due to the internal electric field in the heme cavity. Our results suggest that the conformational transition from the native state, III, into a thermally activated intermediate state, III h, does not substantially affect the internal electric field and causes only moderate rearrangements of the heme pocket, which involves changes, rather than a rupture, of the Fe (3+)-M80 linkage. In the unfolded state, as well as in the alkaline states IV and V, the band splitting is practically eliminated, but the positive Cotton effect observed for the B-band suggests that the proximal environment, encompassing H18 and the two cysteine residues 14 and 17, is most likely still intact and covalently bound to the heme chromophore. Both alkaline states IV and V were found to melt via intermediate states. Unfolded states probed at neutral and alkaline pH can be discriminated, owing to the different intensities of the Cotton bands of the respective B-band transitions. Differences between the ECD intensities of the B-bands of the different unfolded states and alkaline states most likely reflect different degrees of openness of the corresponding heme crevice.

Influence of solvent conditions on refolding of bovine serum albumin

The effect of solvent conditions on the refolding of bovine serum albumin was studied. The rate and extent of refolding was affected by the type of monovalent salt used in the medium. While NaCl and NaBr promoted refolding, NaClO 4 and NaSCN decreased the rate and extent of refolding at 0.2 M concentration. In this respect the relative order in which various anions influenced the refolding process followed the lyotropic series Cl −, Br −, I −, ClO 4 −, SCN −. Urea exhibited two opposite effects on the refolding of albumin: whereas at low concentrations urea increased the extent of refolding, at concentrations above 2.0 M the rate and extent of refolding were dramatically decreased. Addition of ethanol to the medium greatly decreased the refolding even at concentrations as low as 4% (v / v). The effects of these various additives on the refolding behavior of serum albumin is interpreted in tems of subtle changes in the structure of water. It is also shown that, while such changes in the solvent structure affected the rate and extent of refolding, they did not affect the pathway of refolding.