title: Analysis of the pH-dependent thermodynamic stability, local motions, and microsecond folding kinetics of carbonmonoxycytochrome c.
abstract: This paper analyzes the effect of pH on thermodynamic stability, low-frequency local motions and microsecond folding kinetics of carbonmonoxycytochrome c (Cyt-CO) all across the alkaline pH-unfolding transition of protein. Thermodynamic analysis of urea-induced unfolding transitions of Cyt-CO measured between pH 6 and pH 11.9 reveals that Cyt-CO is maximally stable at pH∼9.5. Dilution of unfolded Cyt-CO into refolding medium forms a native-like compact state (NCO-state), where Fe(2+)-CO interaction persists. Kinetic and thermodynamic parameters measured for slow thermally-driven CO dissociation (NCO→N+CO) and association (N+CO→NCO) reactions between pH 6.5 and pH 13 reveal that the thermal-motions of M80-containing Ω-loop are decreased in subdenaturing limit of alkaline pH. Laser photolysis of Fe(2+)-CO bond in NCO-state triggers the microsecond folding (NCO→N). The microsecond kinetics measured all across the alkaline pH-unfolding transition of Cyt-CO produce rate rollover in the refolding limb of chevron plot, which suggests a glass transition of NCO en route to N. Between pH 7 and pH 11.9, the natural logarithm of the microsecond folding rate varies by < 1.5 units while the natural logarithm of apparent equilibrium constant varies by 11.8 units. This finding indicates that the pH-dependent ionic-interactions greatly affect the global stability of protein but have very small effect on folding kinetics.
authors.affiliation: Centre for Chemical Sciences, School of Basic and Applied Sciences, Central University of Punjab, Bathinda, 151001, India; School of Chemistry and Biochemistry, Thapar Institute of Engineering and Technology University, Patiala, 147004, India. Electronic address: rajeshchem01@gmail.com.
authors.forename: Rajesh
authors.initials: R
authors.lastname: Kumar
mesh_terms: D000818:Animals; D002248:Carbon Monoxide / Q000737:chemistry*; D045304:Cytochromes c / Q000737:chemistry*; D006736:Horses; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D009038:Motion; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013056:Spectrophotometry, Ultraviolet; D055672:Static Electricity; D013696:Temperature; D013816:Thermodynamics; D014508:Urea / Q000737:chemistry
chemical_list: D002248:Carbon Monoxide; D014508:Urea; D045304:Cytochromes c
keywords: Ionic interactions; Low-frequency local motion; Microsecond folding kinetics; Thermodynamic stability