J Michael Sauder - Kinetic mechanism of folding and unfolding of Rhodobacter capsulatus cytochrome c2

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      Publication Details (including relevant citation   information):

      Biochemistry (1996) 35: 16852-16862.

      Sauder JM, MacKenzie NE, Roder H


      In spite of marginal sequence homology, cytochrome c2 from    photosynthetic bacteria and the mitochondrial cytochromes c   exhibit some  striking structural similarities, including   the tertiary arrangement of  the three main helices. To   compare the folding mechanisms for these two  distantly   related groups of proteins, equilibrium and kinetic    measurements of the folding/unfolding reaction of cytochrome c2   from  Rhodobacter capsulatus were performed as a function of   guanidine  hydrochloride (GuHCl) concentration in the   absence and presence of a  stabilizing salt, sodium sulfate.   Quenching of the fluorescence of Trp67  by the heme was used   as a conformational probe. Kinetic complexities  due to   non-native histidine ligation are avoided, since cytochrome   c2  contains only one histidine, His17, which forms the   axial heme ligand  under native and denaturing conditions.   Quantitative kinetic modeling  showed that both equilibrium   and kinetic results are consistent with a  minimal   four-state mechanism with two sequential intermediates. The    observation of a large decrease in fluorescence during the   2-ms  dead-time of the stopped-flow measurement (burst   phase) at low GuHCl  concentration, followed by a sigmoidal   recovery of the initial amplitude  toward the unfolding   transition region, is attributed to a  well-populated   compact folding intermediate in rapid exchange with    unfolded molecules. A nearly denaturant-independent process at   low GuHCl  concentrations reflects the rate-limiting   conversion of a compact  intermediate to the native state.   At high GuHCl concentrations, a  process with little   denaturant dependence is attributed to the  rate-limiting   Met96-iron deligation process during unfolding, which is    supported by the kinetics of imidazole binding. The strong    GuHCl-dependence of folding and unfolding rates near the midpoint   of the  equilibrium transition is attributed to   destabilization of each  intermediate and their transition   states in folding and unfolding.  Addition of sodium sulfate   shifts the rate profile to higher denaturant  concentration,   which can be understood in terms of the relative    stabilizing effect of the salt on partially and fully folded   states.

      Address (URL): http://pubs.acs.org/doi/abs/10.1021/bi961976k