Analysis of the pH-dependent thermodynamic stability, local motions, and microsecond folding kinetics of carbonmonoxycytochrome c

作者：Kumar Rajesh^*

来源：Archives of Biochemistry and Biophysics, 2016, 606: 16-25.

DOI：10.1016/j.abb.2016.07.010

• Thermodynamic stability
• Low-frequency local motion
• Microsecond folding kinetics
• Ionic interactions

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 similar to 9.5. Dilution of unfolded Cyt-CO into refolding medium forms a native-like compact state (NCO-state), where Fe2+-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 Omega-loop are decreased in subdenaturing limit of alkaline pH. Laser photolysis of Fe2+-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.

• 出版日期2016-9-15