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-formation of native-like 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 alpha-helical segment (residue 60). The results show that within the first approximately 100 ms of refolding all of the unfolded protein collapses to native-like folding intermediates that contain both antibody binding sites. All three absorbance/fluorescence-detected kinetic phases in the folding of cytochrome c (k1 approximately 5 s-1, k2 approximately 0.4 s-1, k3 approximately 0.03 s-1) are 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 resolved optically-detected refolding phases. Kinetically unresolved folding processes account for 79% and 19% of the total fluorescence change and absorbance change, respectively, observed in equilibrium unfolding. Thus, kinetically unresolved folding reactions appear to be responsible for re-formation of the MAb binding sites within partially folded intermediate species. These species are non-native (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 absorbance-detected slow refolding phase has been measured for folding intermediate(s) of cytochrome c complexed with antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)
Allen
M J
MJ
Department of Biochemistry, University of Texas Health Science Center, San Antonio 78284-7760.
Jemmerson
R
R
Nall
B T
BT
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D003574:Cytochrome c Group / Q000737:chemistry / Q000276:immunology*; D000939:Epitopes; D006736:Horses; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D003574:Cytochrome c Group; D000939:Epitopes