Complexity of aromatic ring-flip motions in proteins: Y97 ring dynamics in cytochrome c observed by cross-relaxation suppressed exchange NMR spectroscopy.

Dynamics of large-amplitude conformational motions in proteins are complex and less understood, although these processes are intimately associated with structure, folding, stability, and function of proteins. Here, we use a large set of spectra obtained by cross-relaxation suppressed exchange NMR spectroscopy (EXSY) to study the 180 degrees flipping motion of the Y97 ring of horse ferricytochrome c as a function of near-physiological temperature in the 288-308 K range. With rising temperature, the ring-flip rate constant makes a continuous transition from Arrhenius to anti-Arrhenius behavior through a narrow Arrhenius-like zone. This behavior is seen not only for the native state of the protein, but also for native-like states generated by adding subdenaturing amounts of guanidine deuterochloride (GdnDCl). Moderately destabilizing concentrations of the denaturant (1.5 M GdnDCl) completely removes the Arrhenius-like feature from the temperature window employed. The Arrhenius to anti-Arrhenius transition can be explained by the heat capacity model where temperature strengthens ground state interactions, perhaps hydrophobic in nature. The effect of the denaturant may appear to arise from direct protein-denaturant interactions that are structure-stabilizing under subdenaturing conditions. The temperature distribution of rate constants under different stability conditions also suggests that the prefactor in Arrhenius-like relations is temperature dependent. Although the use of the transition state theory (TST) offers several challenges associated with data interpretation, the present results and a consideration of others published earlier provide evidence for complexity of ring-flip dynamics in proteins.