Polyols, increasing global stability of cytochrome c, destabilize the thermal unfolding intermediate

Abstract

Studies on the intermediate states of proteins provide essential information on folding pathway and energy landscape of proteins. Osmolytes, known to alter the stability of proteins, might also affect the structure and energy states of folding intermediates. This was examined using cytochrome c (Cyt) as a model protein which forms a spectroscopically detectable intermediate during thermal denaturation transition. Most of the secondary structure and the native heme-ligation were intact in the intermediate state of the protein. Denaturants, urea and guanidinium hydrochloride, and ionic salt destabilizes the intermediate and drive the protein to follow two-state transition. The effect of polyol class of osmolytes, glycol, glycerol, erythritol, xylitol and sorbitol (with OH-groups two to six), on the intermediate was studied using Soret absorbance and far-UV circular dichroism. With the increasing concentration of any of the polyols, the transition-midpoint temperature (T_m) and the enthalpy change (ΔH) for native to intermediate transition were decreased. This indicated that the intermediate was destabilized by the polyols. However, the polyols increased the overall stability of the protein by increasing T_m and ΔH for intermediate to unfolded transition, except for glycol which destabilized the protein. These results show that the polyols could alter the energy state of the intermediate, and the effect of lower and higher polyols might be different on the stability and folding pathway of the protein.

Communicated by Ramaswamy H. Sarma

Keywords:

• Thermal unfolding intermediate
• cytochrome c
• ethylene glycol
• polyols
• protein stability

Acknowledgements

We thank SERB and CSIR for financial support. We acknowledge the instruments facility supported by DST-FIST and UGC-SAP-DRS-I.

Disclosure statement

The authors declare no conflicts of interest.

Additional information

Funding

This work was supported by Council of Scientific and Industrial Research (CSIR) (01-2845/16/EMR-II), India; Department of Science and Technology, India (DST-FIST) (SR/FST/LSI-530/2012); Science and Engineering Research Board (SERB) (EMR/2016/003411); University Grants Commission (UGC-SAP-DRS-I) (5-15/2016/DRS-I).