Rapid folding with and without populated intermediates in the homologous four-helix proteins Im7 and Im9

The kinetics and thermodynamics of the folding of the homologous four-helix proteins Im7 and lm9 have been characterised at PH 7.0 and 10 degrees C. T hese proteins are 60% identical in sequence and have the same three-dimensi onal structure, yet appear to fold by different kinetic mechanisms. The log arithm of the folding and unfolding rates of Im9 change linearly as a funct ion of urea concentration and fit well to an equation describing a two-stat e mechanism (with a folding rate of 1500 s(-1) an unfolding rate of 0.01 s( -1), and a highly compact transition state that has similar to 95% of the n ative surface area buried). By contrast, there is clear evidence for the po pulation of an intermediate during the refolding of Im7, as indicated by a change in the urea dependence of the folding rate and the presence of a sig nificant burst phase amplitude in the refolding kinetics. Under stabilising conditions (0.25 M Na2SO4, pH 7.0 and 10 degrees C) the folding of Im9 rem ains two-state, whilst under similar conditions (0.4 M Na2SO4, pH 7.0 and 1 0 degrees C) the intermediate populated during Im7 refolding is significant ly stabilised (K-UI=125). Equilibrium denaturation experiments, under the c onditions used in the kinetic measurements, show that Im7 is significantly less stable than Im9 (Delta Delta G 9.3 kJ/mol) and the Delta G and m value s determined accord with those obtained from the fit to the kinetic data. T he results show, therefore, that the population of an intermediate in the r efolding of the immunity protein structure is defined by the precise amino acid sequence rather than the global stability of the protein. We discuss t he possibility that the intermediate of Im7 is populated due to differences in helix propensity in Im7 and Im9 and the relevance of these data to the folding of helical proteins in general. (C) 1999 Academic Press.