Defining folding and unfolding reactions of apocytochrome b5 using equilibrium and kinetic fluorescence measurements.
The refolding and unfolding kinetics of a soluble domain of apocytochrome b5 extending from residue 1 to 104 have been characterized using stopped flow and equilibrium-based fluorescence methods. The isolated apoprotein unfolds reversibly in the presence of GuHCl. From cooperative unfolding curves, the conformational stability (Delta G(uw)), in the absence of denaturant, is estimated to be 11.6 +/- 1.5 kJ mol-1 at 10 degrees C. The stability of apocytochrome b5 is lower than that of the corresponding form of the holoprotein (Delta G approximately 25 kJ mol-1) and exhibits a transition midpoint at 1.6 M GuHCl. Kinetic studies support the concept of a two-state model with both unfolding and refolding rates showing an exponential dependence on denaturant concentration with no evidence of the formation of transient intermediates in either limb of the chevron plot. Apocytochrome b5 is therefore an example of a protein in which both kinetics and equilibria associated with folding are described by a two-state model. The values of mku and mkf obtained from kinetic analysis are an indication of a transition state (mku/meq of 0.29) that resembles the native form by retaining similar solvent accessibility and many of the noncovalent interactions found in the apoprotein. The changes in heat capacity support a transition state that resembles the apoprotein with a value for Delta Cpf of -3.6 kJ mol-1 K-1 estimated for the refolding reaction. From these measurements, a model of refolding that involves the rapid nucleation of hydrophobic residues around Trp26 is suggested as a major event in the formation of the native apoprotein.
Structural Biochemistry, Molecular & Cellular Biology, Division of Biomedical Sciences, Queen Mary & Westfield College, London, U.K.
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D000818:Animals; D001059:Apoproteins / Q000737:chemistry* / Q000378:metabolism*; D002417:Cattle; D003573:Cytochrome b Group / Q000737:chemistry* / Q000378:metabolism*; D045303:Cytochromes b; D004795:Enzyme Stability; D019791:Guanidine; D007700:Kinetics; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics
D001059:Apoproteins; D003573:Cytochrome b Group; D045303:Cytochromes b; D019791:Guanidine