Cytochrome c folds through a smooth funnel

A dominant feature of folding of cytochrome c is the presence of nonnative His-heme kinetic traps, which either pre-exist in the unfolded protein or a re formed soon after initiation of folding. The kinetically trapped species can constitute the majority of folding species, and their breakdown Limits the rate of folding to the native state. A temperature jump (T-jump) relax ation technique has been used to compare the unfolding/folding kinetics of yeast iso-2 cytochrome c and a genetically engineered double mutant that la cks His-heme kinetic traps, H33N,H39K iso-2. The results show that the ther modynamic properties of the transition states are very similar. A single re laxation time tau(obs) is observed for both proteins by absorbance changes at 287 nm, a measure of solvent exclusion from aromatic residues. At temper atures near T-m, the midpoint of the thermal unfolding transitions, tau(obs ) is four to eight times faster for H33N,H39K iso-2 (tau(obs) similar to 4- 10 ms) than for iso-2 (tau(obs) similar to 20-30 ms). T-jumps show that the re are no kinetically unresolved (tau < 1-3 mu s T-jump dead time) "burst" phases for either protein. Using a two-state model, the folding (k(f)) and unfolding (k(u)) rate constants and the thermodynamic activation parameters Delta G(f)(double dagger), Delta G(u)(double dagger), Delta H-f(double dag ger), Delta H-u(double dagger), Delta S-f(double dagger), Delta S-u(double dagger) are evaluated by fitting the data to a function describing the temp erature dependence of the apparent rate constant k(obs) (= tau(obs)(-1)) = k(f) + k(u). The results show that there is a small activation enthalpy for folding, suggesting that the barrier to folding is largely entropic. In th e "new view," a purely entropic kinetic barrier to folding is consistent wi th a smooth funnel folding landscape.