RAPID REFOLDING OF NATIVE EPITOPES ON THE SURFACE OF CYTOCHROME-C

Refolding of surface epitopes on horse cytochrome c has been measured by monoclonal antibody binding. Two antibodies were used to probe re-f ormation of nativelike surface structure: one antibody (2B5) binds to native cytochrome c near a type II turn (residue 44) while the other ( 5F8) binds to a different epitope on the opposite face of the protein near the amino terminus of an a-helical segment (residue 60). The resu lts show that within the first approximately 100 ms of refolding all o f the unfolded protein collapses to nativelike folding intermediates t hat contain both antibody binding sites. All three absorbance/fluoresc ence-detected kinetic phases in the folding of cytochrome c (k1 is sim ilar to 5 s-1, k2 is similar to 0.4 s-1, k3 is similar to 0.03 s-1) ar e slower than the rates of re-formation of the antibody binding sites (k(obs) > 10.0 s-1), suggesting that the formation of antibody binding sites precedes the refolding reactions observed in kinetically resolv ed optically-detected refolding phases. Kinetically unresolved folding processes account for 79% and 19% of the total fluorescence change an d absorbance change, respectively, observed in equilibrium unfolding. Thus, kinetically unresolved folding reactions appear to be responsibl e for re-formation of the MAb binding sites within partially folded in termediate species. These species are nonnative (incompletely folded) in that their optical properties are in between those of the unfolded and the fully folded protein. As a test of whether antibody binding to folding intermediate(s) perturbs further folding, the rate of the abs orbance-detected slow refolding phase has been measured for folding in termediate(s) of cytochrome c complexed with antibodies. Neither antib ody had a significant effect on the rate of the absorbance-detected sl ow folding reaction, suggesting that changes in protein conformation a ssociated with the absorbance-detected slow folding phase are not affe cted by possible antibody-induced changes in structure or stabilizatio n of the epitopes. Since epitopes of protein antigens cover an extensi ve surface area (700-800 angstrom2), the results show the major aspect s of the nativelike surface topography are formed early (<100 ms) in f olding intermediates.