STOPPED-FLOW ANALYSIS OF THE REFOLDING OF HEN EGG-WHITE RIBOFLAVIN BINDING-PROTEIN IN ITS NATIVE AND DEPHOSPHORYLATED FORMS

In earlier work (D.A. McClelland, S.H. McLaughlin, R.B. Freedman, and N.C. Price, Biochem. J. 311, 133-137 (1995)), we had shown that during the refolding of hen egg white riboflavin binding protein (RfBP) afte r denaturation in 6M guanidinium chloride, most of the native properti es of the protein are regained within 10-15s of the dilution of the de naturing agent. We have now employed stopped-flow measurements of CD, protein fluorescence and regain of riboflavin binding ability to exami ne the rapid phases of the refolding process. Essentially, all of the native secondary structure as judged by the CD signal at 230nm was reg ained within the dead-time of the instrument in CD mode (approximate t o 8 ms). 80% of the native protein fluorescence was regained within th e dead-time of the instrument in fluorescence mode (1.7 ms). A further 10% was regained with a half time of 30 ms in the case of the apo-pro tein, though the half time was approximately doubled in the presence o f riboflavin. This second phase corresponded with the regain of ribofl avin binding ability. Two slow phases, with half-times of 46s and 1 h involved the regain of the final 10% of fluorescence signal. Binding o f the fluorescent probe, 1-anilino-8-naphthalenesulphonate (ANS) prece ded the formation of the riboflavin binding site. Dephosphorylation of RfBP by treatment with acid phospha:ase did not affect the binding of riboflavin, nor did it alter the kinetics of the refolding process. T his is consistent with the proposal that in vivo phosphorylation occur s on a surface-exposed portion of the protein after the major portion of the folding process is complete. (C) 1998 Elsevier Science B.V.