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 are 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) relaxation technique has been used to compare the unfolding/folding kinetics of yeast iso-2 cytochrome c and a genetically engineered double mutant that lacks His-heme kinetic traps, H33N,H39K iso-2. The results show that the thermodynamic properties of the transition states are very similar. A single relaxation time tau(obs) is observed for both proteins by absorbance changes at 287 nm, a measure of solvent exclusion from aromatic residues. At temperatures near Tm, the midpoint of the thermal unfolding transitions, tau(obs) is four to eight times faster for H33N,H39K iso-2 (tau(obs) approximately 4-10 ms) than for iso-2 (tau(obs) approximately 20-30 ms). T-jumps show that there are no kinetically unresolved (tau < 1-3 micros 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 standard deltaGf, standard deltaGu, standard deltaHf, standard deltaHu, standard deltaSf, standard deltaSu are evaluated by fitting the data to a function describing the temperature 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 the "new view," a purely entropic kinetic barrier to folding is consistent with a smooth funnel folding landscape. Study holds ProTherm entries: 8053, 8054 Extra Details:
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:35 p.m.
|Number of data points||6|
|Proteins||Cytochrome c iso-2 ; Cytochrome c mitochondrial import factor CYC2|
|Assays/Quantities/Protocols||Experimental Assay: dCp ; Experimental Assay: Tm ; Experimental Assay: dHvH|
|Libraries||Mutations for sequence AKESTGFKPGSAKKGATLFKTRCQQCHTIEEGGPNKVGPNLHGIFGRHSGQVKGYSYTDANINKNVKWDEDSMSEYLTNPKKYIPGTKMAFAGLKKEKDRNDLITYMTKAAK|