Global analysis of the effects of temperature and denaturant on the folding and unfolding kinetics of the N-terminal domain of the protein L9

The folding and unfolding kinetics of the N-terminal domain of the ribosoma l protein L9 have been measured at temperatures between 7 and 85 degrees C and between 0 and SM guanidine deuterium chloride. Stopped-flow fluorescenc e was used to measure rates below 55 degrees C and NMR lineshape analysis w as used above 55 degrees C. The amplitudes and rate profiles of the stopped -flow fluorescence experiments are consistent with a two-state folding mech anism, and plots of ln(k) versus guanidine deuterium chloride concentration show the classic v-shape indicative of two-state folding. There is no roll over in the plots when the experiments are repeated in the presence of 400 mM sodium sulfate. Temperature and denaturant effects were fit simultaneou sly to the simple model k = D exp(-Delta G double dagger/RT) where Delta G double dagger represents the change in apparent free energy between the tra nsition state and the folded or unfolded state and D represents the maximum possible folding speed. Delta G double dagger is assumed to vary linearly with denaturant concentration and the Gibbs-Helmholtz equation is used to m odel stability changes with temperature. Approximately 60% of the surface a rea buried upon folding is buried in the transition state as evidenced by c hanges in the heat capacity and III value between the unfolded state and th e transition state. The equilibrium thermodynamic parameters, Delta C(p)deg rees, m and Delta G degrees, all agree with the values calculated from the kinetic experiments, providing additional evidence that folding is two-stat e. The folding rates at 0 M guanidine hydrochloride show a non-Arrhenius te mperature dependence typical of globular proteins. When the folding rates a re examined along constant Delta G degrees/T contours they display an Arrhe nius temperature dependence with a slope of -8600 K. This indicates that fo r this system, the non-Arrhenius temperature dependence of folding can be a ccounted for by the anomalous temperature dependence of the interactions wh ich stabilize proteins. (C) 1998 Academic Press.