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) after denaturation in 6 M guanidinium chloride, most of the native properties of the protein are regained within 10-15 s of the dilution of the denaturing agent. We have now employed stopped-flow measurements of CD, protein fluorescence and regain of riboflavin binding ability to examine the rapid phases of the refolding process. Essentially, all of the native secondary structure as judged by the CD signal at 230 nm was regained within the dead-time of the instrument in CD mode (approximately 8 ms). 80% of the native protein fluorescence was regained within the 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-protein, though the half time was approximately doubled in the presence of riboflavin. This second phase corresponded with the regain of riboflavin binding ability. Two slow phases, with half-times of 46 s and 1 h involved the regain of the final 10% of fluorescence signal. Binding of the fluorescent probe, 1-anilino-8-naphthalenesulphonate (ANS) preceded the formation of the riboflavin binding site. Dephosphorylation of RfBP by treatment with acid phosphatase did not affect the binding of riboflavin, nor did it alter the kinetics of the refolding process. This is consistent with the proposal that in vivo phosphorylation occurs on a surface-exposed portion of the protein after the major portion of the folding process is complete.
McClelland
D A
DA
Department of Biological and Molecular Sciences, University of Stirling, UK.
Price
N C
NC
D000818:Animals; D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D002645:Chickens; D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004529:Egg White; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D007700:Kinetics; D026901:Membrane Transport Proteins; D010766:Phosphorylation; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D012256:Riboflavin / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D001059:Apoproteins; D002352:Carrier Proteins; D026901:Membrane Transport Proteins; C019079:riboflavin-binding protein; D012256:Riboflavin