Structural, kinetic and thermodynamic characterizations of SDS-induced molten globule state of a highly negatively charged cytochrome c

• Published in: Journal of biochemistry (Tokyo) 2019-02, Vol.165 (2), p.125-137
• Main Authors: Jain, Rishu, Sharma, Deepak, Kumar, Rakesh, Kumar, Rajesh
• Format: Article
• Language: English
• Subjects: Cytochromes c - chemistry; Cytochromes c - metabolism; Kinetics; Protein Denaturation; Sodium Chloride - chemistry; Sodium Dodecyl Sulfate - chemistry; Thermodynamics
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/jb/mvy087

Abstract This study presents the structural, kinetic and thermodynamic characterizations of previously unknown submicellar concentrations of SDS-induced molten globule (MGSDS) state of a highly negatively charged base-denatured ferricytochrome c (UB-state) at pH ∼12.8 (±0.2). The far-UV CD, near-UV CD, ANS-fluorescence data of UB-state in the presence of different concentrations of SDS indicate that the submicellar concentrations of SDS (≤0.4 mM) transform the UB-state to MGSDS-state. The MGSDS-state has native-like α-helical secondary structure but lacks tertiary structure. The free energy change (ΔG°D) for UB→ MGSDS transition determined by far-UV CD (∼2.7 kcal mol−1) is slightly higher than those determined by fluorescence (∼2.0 kcal mol−1) at 25°C. At very low SDS and NaCl concentrations, the MGSDS-state undergoes cold denaturation. As SDS concentration is increased, the thermal denaturation temperature increases and the cold denaturation temperature decrease. Kinetic experiments involving the measurement of the CO-association rate to the base-denatured ferrocytochrome c at pH ≈12.8 (±0.2), 25°C indicate that the submicellar concentrations of SDS restrict the internal dynamics of base-denatured protein.