Analysis of Folding and Unfolding Reactions of Cytochrome b5

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Abstract
The guanidine hydrochloride- (GuHCl-) induced unfolding and refolding of a recombinant domain of bovine microsomal cytochrome b5 containing the first 104 amino acid residues has been characterized by both transient and equilibrium spectrophotometric methods. The soluble domain is reversibly unfolded and the equilibrium reaction may be monitored by changes in absorbance and fluorescence that accompany denaturation of the native protein. Both probes reveal a single cooperative transition with a midpoint at 3 M GuHCl and lead to a value for the protein stability (ΔGuw) of 26.5 kJ mol-1. This stability is much higher than that reported for the corresponding form of the apoprotein (∼7 kJ mol-1). Transient changes in fluorescence and absorbance during protein unfolding exhibit biphasic profiles. A fast phase occupying ∼30% of the total amplitude is observed at high denaturant concentrations and becomes the dominant process within the transition region. The rates associated with each process show a linear dependency on GuHCl concentration, and at zero denaturant concentration the unfolding rates (kuw) are 4.5 × 10-5 s-1 and 5.2 × 10-6 s-1 at 25 °C. The pattern of unfolding is not correlated with covalent heterogeneity, since a wide range of variants and site-directed mutants exhibit identical profiles, nor is the unfolding correlated with cis−trans Pro isomerization in the native state. In comparison with the apo form of cytochrome b5, the kinetics of refolding and unfolding are more complex and exhibit very different transition states. The data support a model for unfolding in which heme−protein interactions give rise to two discernible rates of unfolding. From an analysis of the activation parameters associated with each process it is established that two structurally similar transition states differing by less than 5 kJ mol-1 exist in the unfolding reaction. Protein refolding exhibits monophasic kinetics but with distinct curvature apparent in plots of ln kobs versus denaturant concentration. The data are interpreted in terms of alternative routes for protein folding in which a “fast track” leads to the rapid ordering of structure around Trp26 for refolding while a slower route requires additional reorganization around the hydrophobic core.