On the relationship between protein stability and folding kinetics: A comparative study of the N-terminal domains of RNase HI, E-coli and Bacillus stearothermophilus L9

There is currently a great deal of interest in proteins that fold in a sing le highly cooperative step. Particular attention has been focused on elucid ating the factors that govern folding rates of simple proteins. Recently, t he topology of the native state has been proposed to be the most important determinant of their folding rates. Here we report a comparative study of t he folding of three topologically equivalent proteins that adapt a particul arly simple alpha/beta fold. The folding kinetics of the N-terminal domain of RNase HI and the N-terminal domain of the ribosomal protein L9 from Esch erichia coli (eNTL9) were compared to the previously characterized N-termin al domain of L9 from Bacillus stearothermophilus (bNTL9). This 6.2 kDa prot ein, which is one of simplest examples of the ABC alphaD motif, folds via a two-state mechanism on the millisecond to submillisecond time scale. The R Nase HI domain and bNTL9 have very similar tertiary structures but there is little similarity in primary sequence. bNTL9 and eNTL9 share the same biol ogical function and a similar primary sequence but differ significantly in stability. Fluorescence-detected stopped-flow experiments showed that the t hree proteins fold in a two-state fashion. The folding rates in the absence of denaturant were found to be very different, ranging form 21 s(-1) to 79 0 s(-1) at 10 degreesC. The diverse folding rates appear to reflect large d ifferences in the stability of the proteins. When compared at an isostabili ty point, the folding rates converged to a similar value and there is a str ong linear correlation between the log of the folding rate and stability fo r this set of proteins. These observations are consistent with the idea tha t stability can play an important role in dictating relative folding rates among topologically equivalent proteins. (C) 2001 Academic Press.