Slow unfolding and refolding kinetics of the mesophilic Rop wild-type protein in the transition range

We describe the guanidinium hydrochloride induced folding kinetics of the f our-helix-bundle protein Pop wild-type (wt) under equilibrium conditions at three temperatures. The choice of appropriate denaturant conditions inside the transition range permitted, in combination with equilibrium transition curves, the determination of both unfolding and refolding rate constants. The ratio of the rate constants at zero denaturant concentration provided e quilibrium constants and standard free energy changes that are in good agre ement with values obtained in previous differential scanning calorimetry st udies. The Delta G(D)(0) values for 19, 25 and 40 degrees C calculated from the present kinetic studies are, respectively, 66.8, 70.8 and 57.2 kJ . mo l(-1). The unfolding reactions are extremely slow under these conditions. E quilibrium was reached only after 18, 12 and 6 days at 19, 25 and 40 degree s C. These results demonstrate that for Pop wt high stability correlates wi th slow folding kinetics.