Denatured states of yeast cytochromecinduced by heat and guanidinium chloride are structurally and thermodynamically different

Abstract

A sequence alignment of mammalian cytochromes c with yeast iso-1-cytochrome c (y-cyt-c) shows that the yeast protein contains five extra N-terminal residues. We have been interested in understanding the question: What is the role of these five extra N-terminal residues in folding and stability of the protein? To answer this question we have prepared five deletants of y-cyt-c by sequentially removing these extra residues. During our studies on the wild type (WT) protein and its deletants, we observed that the amount of secondary structure in the guanidinium chloride (GdmCl)-induced denatured (D) state of each protein is different from that of the heat-induced denatured (H) state. This finding is confirmed by the observation of an additional cooperative transition curve of optical properties between H and D states on the addition of different concentrations of GdmCl to the already heat denatured WT y-cyt-c and its deletants at pH 6.0 and 68°C. For each protein, analysis of transition curves representing processes, native (N) state ↔ D state, N state ↔ H state, and H state ↔ D state, was done to obtain Gibbs free energy changes associated with all the three processes. This analysis showed that, for each protein, thermodynamic cycle accommodates Gibbs free energies associated with transitions between N and D states, N and H states, and H and D states, the characteristics required for a thermodynamic function. All these experimental observations have been supported by our molecular dynamics simulation studies.

Thermally versus Chemically Denatured Protein States

“…As an additional example, we show the thermal and chemical unfolding curve of PurR, a member of the LacI DNA-binding domain family. Clear differences in the CD signals of the denatured states are also apparent in these data (Figure 1C,D), consistent with earlier works 9–20…”

“…Thermal perturbations of urea or GuHCl-denatured states of folded proteins reveal a continuous and reversible increase in the intensity of the CD signal at 222 nm (Figure A and Figures S3 and S4). ,,− ,− Even IDPs exhibit a tendency where the signal at 222 nm displays a negative slope with temperature (Figure B). − Interestingly, the CD signal range and slopes (34.7 ± 9.4 deg cm 2 dmol –1 K –1 ) are very similar despite the large differences in sequence. The CD signals at high temperatures and in the absence of denaturant (U T ) agree very well with the CD signals at high denaturant concentration and high temperatures (U D, High T ) with a mean absolute difference of just ∼550 deg cm 2 dmol –1 (Figure C) for a small database of proteins (Table S2).…”

“…Thermal perturbations of urea or GuHCl-denatured states of folded proteins reveal a continuous and reversible increase in the intensity of the CD signal at 222 nm (Figure 3A and Figures S3 and S4). 10,11,13–15,17–20 Even IDPs exhibit a tendency where the signal at 222 nm displays a negative slope with temperature (Figure 3B). 26–29 Interestingly, the CD signal range and slopes (34.7 ± 9.4 deg cm 2 dmol −1 K −1 ) are very similar despite the large differences in sequence.…”

“…The simplest spectroscopic technique used to study denaturation is far-ultraviolet (far-UV) circular dichroism (hereafter termed CD) that interrogates the local conformational preference of peptide bonds 8. Earlier studies have pointed to differences in the magnitude of the CD signal between thermally and chemically denatured states on specific proteins 9–20. Here, we extend these works by collecting CD data from the literature, show that the differences observed previously are robust to structure−sequence changes, and explain their implications.…”

Structural basis of urea-induced unfolding: Unraveling the folding pathway of hemochromatosis factor E

“…The stability of multi-domain proteins can be estimated by the equilibrium unfolding studies in the presence of denaturants, urea or GdmCl which may induces different unfolding pathways [53][54][55]. HFE is a major iron-regulatory protein involved in the ironregulation and is also associated with cancer [56] and Alzheimer diseases [57].…”