Indexed on: 11 Sep '18Published on: 11 Sep '18Published in: Biochemistry
The folding of many globular proteins from the unfolded (U) to the native (N) state appears to be describable by a two-state N↔U model, which has led to the general belief that protein folding occurs in a highly cooperative manner. One reason for the widespread belief in "two-state folding" is that protein folding reactions are invariably studied by ensemble averaging probes, and not by probes that can distinguish between, as well as quantify the multiple conformations that may be present. Consequently, how cooperativity is affected by protein stability, protein sequence and solvent conditions, is poorly understood. In this study, hydrogen exchange coupled to mass spectrometry (HX-MS) of the PI3K SH3 domain, was carried out in the presence of a stabilizing osmolyte, trimethylamine-N-oxide (TMAO). By showing that HX occurs under the EX1 regime even in the presence of 2 M TMAO, it became possible to examine the temporal evolution of the populations of the different conformations present together. A strong link between protein folding cooperativity and protein stability is revealed: increasing stability is accompanied by an increase in the ruggedness of the free energy landscape, as well as diminished cooperativity: the number of amide sites simultaneously opening up their structure decreases with increasing TMAO concentration. A comparison of the effect of TMAO to that of urea on the intrinsic dynamics of the PI3K SH3 domain indicates that TMAO not only counteracts the effect of urea on protein stability but also on protein folding cooperativity.