Thermodynamics of the Thermal Denaturation of Acid Molten Globule State of Cytochrome c Indicate a Reversible High-Temperature Oligomerization Process

Abstract

In this study, we performed differential scanning calorimetry (DSC) and pressure perturbation calorimetry (PPC) analysis of the thermal transition of cytochrome c from an acidic molten globule (MG) state with the protein concentrations of 0.5-18.2 mg/mL. DSC profiles were highly reversible and showed clear protein-concentration dependence, indicating that reversible oligomerization occurred accompanying the thermal transition from the MG state. The DSC and PPC data required at least a six-state model (MG1 ⇄ MG2 ⇄ D ⇄ / I ⇄ / I ⇄ / I) including three new oligomeric states: dimer (I), trimer (I), and tetramer (I) in addition to the three monomeric states previously characterized. Dynamic light scattering confirmed the oligomerization during the thermal transition. The partial specific volumes of these oligomeric states were found to be smaller than those of the monomeric states, MG2 and D, indicating dehydration of hydrophobic surface or hydration of released anions may occur with the reversible oligomerization.

Reversible Oligomerization and Reverse Hydrophobic Effect Induced by Isoleucine Tags Attached at the C-Terminus of a Simplified BPTI Variant

“…Furthermore, C5I's T m decreased with increasing protein concentrations (Figure 1C). We observed a similar situation for Dengue 4 ED3 9 and cytochrome c, 14 where the decrease in T m upon an increased protein concentration was caused by the reversible oligomerization of the protein at high temperature in an unfolded or partially unfolded state (a non-native state). We thus decided to analyze the putative presence of an RO state in the present system.…”

“…The thermal transition of C5I was fully reversible and independent of the scanning rate between 20 and 80 K/h (Figure 3B), indicating that its thermodynamics can be analyzed as an equilibrium reaction. Thus, in line with the CD observations, the concentration dependence of the denaturation profile monitored by DSC required some RO states at high temperatures as previously shown in the thermal transition of molten globule state of cytochrome c. 14 On the contrary, in the case of reversible thermal transition of oligomeric proteins in the native state, it is frequently observed that the thermal stability of the native sate increases, namely apparent T m increases, with increasing protein concentration because the oligomers dissociated in the denatured state. 11 The difference between the chemical stoichiometry of N and D state is the origin of protein-concentration dependence of the thermodynamic stability in equilibrium.…”

“…Recently, a peculiar type of oligomerization that associates in a reversible manner has been observed in DSC measurements. ,− In particular, we observed that cytochrome c and the third domain of the envelope protein from dengue virus serotype 4 (DEN-4 ED3) are partially or fully unfolded when they are associated in a reversibly oligomerized (RO) state according to a comparison of their thermal denaturation curves measured by DSC and by far-UV circular dichroism (CD), which monitors the secondary structure content . Thus, the reversibility of the association and the average size remaining constant over a long time make the RO state very similar to quaternary structures formed by native proteins at ambient temperatures, such as that observed in hemoglobin. , Alike the quaternary structures of native proteins are stabilized by specific intermolecular interactions between natively folded proteins, it is reasonable to expect that the quaternary structures dissociate once the individual proteins unfold.…”

Misfolding of a Single Disulfide Bonded Globular Protein into a Low-Solubility Species Conformationally and Biophysically Distinct from the Native One

“…In particular, ANS and Trp fluorescence suggested that the misfolded proteins had molten globule like properties (Figure 3E,F). However, in sharp contrast with the molten globule state [34,35], the misfolded species is not in equilibrium with the native state: once the single intramolecular SS bond is formed it cannot interconvert into the native state. Thus, the misfolded species that is observed here is an off-pathway species, which is again distinct from the molten globule state that is thought to be a folding intermediate leading to the formation of a native state [36,37,38].…”

Thermal analysis of some novel pyrimidine derivatives

“…The analysis is popular in various industries namely pharmaceutical [10,11], forensic [12,13], food [14,15], ceramics [16,17], polymer [18,19], composites [20,21] and semiconductors [22] industries. Further, by thermal analysis, kinetic parameters of thermally simulated reactions can be evaluated which provides a deeper insight in to the mechanism of high energetic compounds [23][24][25][26].…”