The refolding of riboflavin binding protein

Abstract

Hen egg riboflavin binding protein (RfBP) acts as a source of riboflavin to the developing embryo. It is the most abundant vitamin binding protein in the egg white. Mutations giving rise to a lack of RfBP lead to embryo death at approximately 13 days. RfBP binds riboflavin tightly in a 1:1 ratio. On formation of this complex, the fluorescence of riboflavin is completely quenched; this quenching is thought to be due to the stacking of aromatic groups within the hydrophobic binding pocket. This quenching provides a convenient assay for the integrity of the riboflavin-binding site of the protein. RfBP consists of a single polypeptide chain of 219 amino acids of molecular mass 29.2 kDa. RfBP undergoes a number of post-translational modifications, namely: the formation of nine disulphide bonds, extensive glycosylation on Asn 36 and Asn 147, and the phosphorylation of eight serine side chains from between Ser 186 and Ser 197. The unfolding and refolding of RfBP was studied by denaturing in 6M guanidium chloride, followed by dilution in buffer, to start refolding. The processes were followed by both steady-state and stopped-flow circular dichroism and fluorescence spectroscopy. RfBP was found to readily unfold and refold, provided the disulphide bonds were intact. The regain of secondary structure was found to be too rapid to measure by the methods available (<12msec). The regain of tertiary structure was found to consist of 4 main phases, and a large proportion (80%) of the tertiary structure formed within 2 msec. The regain of riboflavin binding ability was complete at the end of the second phase, a reaction with a half-life of around 30 msec. In the presence and absence of riboflavin, the kinetics for the first 3 stages of tertiary structure changes seemed to be identical. In the presence of riboflavin, however, seemed to impede the completion of the final, very slow stage, with the refolding reaction only going to 95% completion. The dephosphorylation of the protein seemed to have no affect on this process. When the 9 disulphide bonds are reduced however, RfBP is unable to spontaneously reoxidise to a native-like state in the presence of an oxidised/reduced glutathione redox system. However, the addition of protein disulphide isomerase to the system increases significantly the yield of successfully reoxidised RfBP to about 50%. Attempts to prepare deglycosylated RfBP by chemical methods were unsuccessful since the treatment led to fragmentation of the polypeptide chain.