Refolding of native and recombinant chicken riboflavin carrier (or binding) protein: evidence for the formation of non-native intermediates during the generation of active protein.
Riboflavin carrier (or binding) protein (RCP) is a phosphoglycoprotein originally purified from the egg white, yolk and serum of laying hens. The 18 cysteine residues present in RCP form nine disulfide bridges, allowing the protein to form a compact structure to generate a hydrophobic pocket in which riboflavin sits. We studied the refolding of totally reduced and denatured egg white RCP and found that the protein initially folded to generate a molecule that did not possess riboflavin-binding activity, despite near-complete oxidation of the cysteine residues. Riboflavin-binding activity was then slowly regained, but the final refolded form of the protein was less compact in structure than the native molecule, due to incomplete oxidation of all the cysteine residues. Denatured and reduced dephosphorylated RCP refolded as efficiently as the native protein, with similar rates of disulfide-bond oxidation and generation of riboflavin binding, showing that the phosphoserine stretch of RCP has little role to play during refolding. In order to study the role of glycosylation in the refolding process, the cDNA for full-length RCP was expressed in Escherichia coli and purified. Recombinant RCP refolded only in the presence of redox buffers, demonstrating that glycosylation of RCP could allow the formation of high yields of productive intermediates in the folding pathway. Using a panel of conformation-specific monoclonal antibodies to RCP, it appeared that the folding intermediates of RCP possessed a structure distinctly different to the native protein, indicating that the correct folding pathway of RCP passed through conformation(s) generated by non-native disulfide bridges.
Pattanaik
P
P
Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore, India.
Sooryanarayana
Adiga
P R
PR
Visweswariah
S S
SS
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D002352:Carrier Proteins / Q000737:chemistry*; D002645:Chickens; D004220:Disulfides / Q000378:metabolism; D004527:Egg Proteins / Q000737:chemistry*; D005978:Glutathione / Q000378:metabolism; D019803:Glutathione Disulfide / Q000378:metabolism; D006031:Glycosylation; D007700:Kinetics; D026901:Membrane Transport Proteins; D010084:Oxidation-Reduction; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000737:chemistry; D012256:Riboflavin / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D000911:Antibodies, Monoclonal; D002352:Carrier Proteins; D004220:Disulfides; D004527:Egg Proteins; D026901:Membrane Transport Proteins; D011994:Recombinant Proteins; C019079:riboflavin-binding protein; D005978:Glutathione; D012256:Riboflavin; D019803:Glutathione Disulfide