Unfolding and refolding of hen egg-white riboflavin binding protein

Unfolding and refolding of hen egg-white riboflavin binding protein Simon Allen, Lewis Stevens, Doris Duncan, Sharon M. Kelly and Nicholas C. Price Department of Biological and Molecular Sciences, Stirling University, Stirting FK9 4LA, UK

(Received 22 May 1992; revised 9 September 1992) The unfolding and refolding of riboflavin-binding protein ( RfBP) from hen egg-white induced by addition of guanidinium chloride ( GdnHC1), and its subsequent removal by dialysis have been studied by e.d. and fluorescence for both the native and reduced protein. The reduction of its nine disulphide bonds causes a reduction in the secondary structure (or-helix plus fl-sheet) from 63% to 33% of the amino acid residues. Unfolding of the native protein occurred in two phases," the first involving a substantial loss of tertiary structure, followed by a second phase involving loss of secondary structure at higher GdnHCl concentrations. By contrast this biphasie behaviour was not discernible in the reduced protein. The loss of ability to bind riboflavin occurred after the first phase of unfolding. Comparison of unfolding of the holoprotein and apoprotein suggested that riboflavin has only a small stabilizing effect on the unfolding process. After removal of GdnHCl, the holoprotein, apoprotein and reduced protein assumed their original conformations. The significance of the results in relation to various models for protein folding is discussed. Keywords: Riboflavin;riboflavinbinding protein; folding;unfolding;denaturation;renaturation;circular dichroism;

fluorescence;egg-white;moltenglobule

Introduction Hen egg-white riboflavin binding protein (RfBP) is involved in supplying the developing embryo with riboflavin. RfBP has an unusual distribution in eggs, being present in both yolk and albumen. RfBP present in the yolk is synthesized in the liver and transported through the blood stream to the developing oocyte, whereas RfBP present in the albumen is synthesized in the oviduct, as are other egg-white proteins. RfBP from the yolk and albumin have identical amino acid sequences and are the products of the same gene, but they differ in their glycosylation patterns 1. RfBP possesses a number of features which make it an interesting system for the study of protein folding and maturation. The protein from hen egg-white is a single polypeptide chain of 219 amino acids whose amino acid sequence has been deduced by direct 2 and indirect (via eDNA 3 ) means. The three-dimensional structure has not been determined, but crystals that diffract to 2.8 A have been obtained 1. Numerous post-translational modifications occur principally: (i) the formation of nine disulphide bonds, (ii) the attachment of oligosaccharides to Asn 36 and Asn 147, and (iii) phosphorylation of eight serine side chains between Ser 186 and Ser 1971. The two proteins containing disulphide bonds that have been most used to study folding and refolding are ribonuctease and bovine pancreatic trypsin inhibitor4. Both are small proteins having 124 and 58 amino acids, respectively. RfBP is larger, and has the advantage that its biological activity can be readily monitored, since the native apoprotein quenches the fluorescence of riboflavin on binding 5. With ribonuclease and bovine pancreatic trypsin inhibitor, the enzyme substrate complex, or the enzyme inhibitor complexes are not easily detected directly, but inferred through the change in enzyme activity. 0141-8130/92/060333-05 © 1992 Butterworth-HeinemannLimited

As part of our studies on the maturation of a number of egg-white proteins we have obtained data on the unfolding (by guanidinium chloride, GdnHCI) and refolding of RfBP, both in the holo- and apoprotein and in the reduced form, in which the disulphide bonds have been cleaved by reduction with NaBH4. Our results show that provided the disulphide bonds remain intact the protein can be refolded with high efficiency. The results also suggest the possibility of an intermediate structure during unfolding which has reduced tertiary structure, but retains most of the secondary structure, the properties expected of the 'molten globule '6. Reduction of the disulphide bonds leads to considerable loss of secondary structure, but the unfolding/refolding cycle does not lead to any change in this reduced level of structure.

Experimental RfBP from hen egg-white was purified by ion exchange chromatography using DEAE-Sephadex A-50, as previously described 2'7. Riboflavin was removed from the holoprotein by ion-exchange chromatography on SESephadex A-50 using a 0.1 to 0.5 M NaC1 gradient at pH 4.18. The disulphide bridges present in RfBP were reduced by NaBH 4 using the procedure of Baker and Panow 9, modified by the omission of DTNB (5,5'dithiobis(2-nitrobenzoic acid ) ). After reduction, RfBP was dialysed against 0.1 M sodium phosphate/lmM dithiothreitol, pH 7.0. The holoprotein, apoprotein and reduced RfBP were unfolded by incubation for 15min at room temperature with GdnHC1 dissolved in 0.1 M sodium phosphate buffer, pH 7.0. The concentrations of GdnHC1 (Ultrapure grade from Gibco-BRL) were checked by refractive index measurements t°. For renaturation studies, the samples containing GdnHC1 were dialysed overnight against two changes of 1000 volumes of 0.1 M sodium phosphate buffer, pH 7.0.

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Folding of riboflavin-binding protein." S. Allen et al. Circular dichroism spectra were recorded at 20°C in a Jasco J-600 spectropolarimeter. Far u.v. (190-260 nm) spectra were recorded at protein concentrations of about 0.05 to 0.1 mg/ml using cells of pathlength 0.05 cm or 0.1 cm. Molar ellipticity values were calculated using a value of 133.3 for the mean residue weight, a value which takes into account the carbohydrate and phosphate group content of the protein 2. The secondary structure content was determined by using the C O N T I N procedure over the range 190-240 nm 11. The protein concentration was determined by the Coomassie Blue binding method1 ~ and spectrophotometrically at 282 nm using the published absorption coefficients for the holo- and apoprotein 13. Fluorescence spectra were recorded at 20°C in a Perkin Elmer LS 50 fluorimeter with excitation at 280 nm at a protein concentration of 0.055/ng/ml. The quenching of fluorescence of riboflavin by the apoprotein-binding protein was measured as emission at 520nm after excitation at 370nm 5. Binding of 20/~M 8-anilino-1naphthalene sulphonic acid (ANS) to the holoprotein and apoprotein ( 6 0 # g / m l ) in the range of GdnHC1 concentrations 0 to 6.0 M was studied by monitoring the fluorescence at 4 7 0 n m after excitation at 380nm. Enhancements were quoted relative to the fluorescence of 20/~M ANS in the same concentrations of GdnHC1. Spectrophotometric titration of the tyrosine residues against K O H was carried out at 295 nm ~4.

Results and discussion The secondary structures of the holoprotein, apoprotein and reduced RfBP were compared by measuring their far u.v.c.d, spectra between 1 9 0 - 2 6 0 n m (Figurel). There is little difference between those of the holo- and apoprotein and this is consistent with previous results 7,13; however the spectrum of RfBP is significantly different. Analysis of the c.d. spectra 11 shows a marked reduction in the proportion of secondary structure from 24% a, 39% fl and 37% remainder in the holoprotein to 22% a, 10% fl and 67% remainder in the reduced RfBP. There are also differences in the fluorescence spectra of the apoprotein and reduced RfBP (Figure 2), the former having an emission maximum at 345-6 nm whereas the latter is at 354 nm. The difference suggests a greater exposure of tryptophan residues to the solvent

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Figure 1 Far u.v.c.d, spectra of reduced RfBP (r), holo-RfBP (h), and apo-RfBP (a). Spectra were recorded at 20°C in 0.1 M sodium phosphate buffer, pH 7.0, with protein concentration of 0.06mg/ml for reduced RfBP (pathlength =0.1cm), 0.1 mg/ml for holo-RfBP and apo-RfBP (pathlength = 0.05 cm)

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Figure 2 Fluorescence spectra of apo-RfBP (a), and reduced RfBP (b). The protein concentration was 0.06 mg/ml, and the excitation wavelength was 280 nm

in the case of the reduced RfBP and is consistent with loss of tertiary structure, since 355 nm corresponds to the emission maximum of N-acetyltryptophan amide aS. The unfolding of the holoprotein, apoprotein and reduced RfBP was studied using GdnHC1 concentrations in the range 0-6.0 M, and the changes in secondary and tertiary structures studied by c.d. and fluorescence spectroscopy, respectively. The )~rnax for fluorescence emission for both the holoprotein and apoprotein increases from 345 nm at 0 M GdnHC1 to 356 nm at 6.0M GdnHC1 with increasing concentrations of GdnHCI, the most pronounced change occurring between 1.0-2.5 M GdnHC1. For concentrations between 2 and 4 u the 2.,,x was in the range 348-350 nm. The changes in fluorescence intensity measured at 350 nm and ellipticity at 225 nm for the holoprotein, the apoprotein and the reduced RfBP are given in Figures 3a, b and c. The changes in fluorescence intensity at 350 nm for the holo- and apoprotein, both showing a marked increase at GdnHC1 concentrations between 1 . 0 u and 2.5 u, followed by a plateau, and then a further increase between 4.0 M and 6.0 M. The reduced RfBP shows a linear increase through the range of GdnHC1 concentrations, but the total increase in fluorescence is smaller. The changes in the ellipticity at 225 nm (a measure of secondary structure) when the holo- and apoproteins are exposed to increasing concentrations of G d n H C l occur in a similar biphasic manner, but the largest proportional increase occurs between 4.0M and 6.0M. In 6.0M GdnHC1 the c.d. spectrum above 205 nm (the lower limit imposed by the absorbance of GdnHC1) is that of a random coil. The reduced RfBP shows a linear increase, but again the total increase is smaller. This is consistent with the smaller proportion of secondary structure initially present (33% in reduced RfBP compared to 63% in the holoprotein, Figure 1 ). These results suggest that loss of much of the tertiary structure of the holo- and apoprotein occurs between 0-2.0 M GdnHC1, and that of the secondary structure between 4.0 M and 6.0 M, with a possible intermediate conformation stable in the plateau region (2.0-4.0 M GdnHC1). A further insight into the tertiary structure of RfBP was obtained by carrying out a spectrophotometric titration of the tyrosine residues in the absence, and in the presence, of 6.0 M GdnHC1. When the holo- and the apoprotein were titrated against K O H , both showed approximately four tyrosine residues having pKap p of 10.6, and five showing PKap p of 12.8. When titrated in the presence of 6.0 M guanidine hydrochloride a single inflection was seen in the titration curve corresponding to pKap p 10.2 10.6 for all nine tyrosine residues. The reversibility of the unfolding process was assessed

Folding of riboflavin-binding protein: S. Allen et al. Table 1 Refolding of hen egg-white RfBP after unfolding in 6.0 M guanidine hydrochloride

~maxemission in 6.0 U GdnHC1 (nm)

2max after dialysis (nm)

% regain of

% regain of

Sample

2max emission (nm)

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Figure 3

Unfolding of holo-RfBP (a), apo-RfBP (b) and

reduced RfBP (c) in GdnHC1. Unfolding was monitored by changes in ellipticity at 225 nm ( O ) and fluorescence at 350 nm ( • ) . Protein concentrations were 0.1 mg/ml for the holo- and apoprotein, and 0.06 mg/ml for the reduced RfBP

Figure 4 The quenching of fluorescence of riboflavin on addition of apo-RfBP. Apo-RfBP was added to solutions containing 2.31 x 10-7M riboflavin dissolved in 0.1 M sodium phosphate buffer, pH 7.0 and the following concentrations of GdnHCh ( 0 ) , 3.0 M; ( + ), 2.5 M; (Q), 2.0 M; (A), 1.5 M; and no GdnHC1 (Zk). Fluorescence emission was measured at 520nm after excitation at 370nm. The % of the initial fluorescence at the equivalence point (protein/riboflavin = 1.0) at each GdnHC1 concentration was 0u, 13%; 1.5 M, 30%; 2.0M, 43%; 2.5N, 72% and 3.0M, 96% by the extent to which the spectral changes are reversed when the GdnHC1 is removed by dialysis. Each sample was incubated with 6.0 M GdnHC1 for 15 min and then dialysed overnight against 0.t M sodium phosphate buffer, p H 7.0. The regain of the native conformation of the holo- and apoproteins, and of the initial conformation of the reduced RfBP was assessed by the regain of ellipticity, intensity of fluorescence, and the '~max of fluorescence emission (Table 1). All three parameters suggest complete reversibility of the unfolding process. It was also found that the ability of the apoprotein to bind riboflavin was completely restored (data not shown). The results given above suggest that the unfolding process induced by GdnHC1 occurs in two stages, and initial loss of tertiary structure followed by a loss of secondary structure at higher concentrations of GdnHC1. The activity of RfBP can be tested by its ability to bind riboflavin, which is accompanied by a quenching of fluorescence emission at 520 rim. Riboflavin, dissolved in a given concentration of buffered GdnHC1 was titrated against the apoprotein in the same concentration of GdnHC1 and the quenching of emission at 520 nm measured. Figure 4 shows the quenching of fluorescence by the apoprotein, in the presence of increasing concentrations of GdnHC1, and the percentage reduction

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Folding of riboflavin-binding protein: S. Allen et al.

in fluorescence at the equivalence point for each data set given. The ability to bind riboflavin decreases sharply at GdnHC1 above 2.0 M. RfBP unfolds completely with increasing concentrations of GdnHC1, so that in spite of having nine disulphide bridges there is complete loss of secondary and tertiary structure. This change occurs in two phases, the first of which involves primarily the loss of tertiary structure, as shown by the fluorescence due to tryptophan residues. A possible intermediate structure may exist at GdnHC1 concentrations between 2.0 M and 4.0 M. Such an intermediate appears unable to bind riboflavin, or is able to bind it only weakly. Riboflavin appears to be able to stabilize RfBP against denaturation to only a limited extent, since both holo- and apoproteins show similar changes in fluorescence intensity. The intermediate observed between 2.0 and 4.0 ~ GdnHC1 appears to have retained a substantial amount of secondary structure, but has lost much of its tertiary structure as judged by fluorescence and has lost its ability to bind riboflavin. Although these characteristics might indicate that the intermediate resembles a 'molten globule', this conclusion was not supported by studies of the binding of ANS which has been proposed as a test for this state t6. Over the range of GdnHC1 concentrations from 0 to 6.0 M the enhancement of ANS fluorescence in the presence of either holoprotein or apoprotein declined steadily from 2.6-fold (in the absence of GdnHC1) to 0.95-fold (6.0 M GdnHC1). It is therefore possible that the intermediate represents a state in which the unfolding of two (or more) domains occurs independently. At present no evidence for such domains has been reported from studies of proteolysis or thermal denaturation of the protein. In the absence of the disulphide bridges, the reduced RfBP is able to refold and assume its initial conformation. The study of the folding/unfolding of proteins in vitro can help to elucidate the folding process in vivo 4. The intermediate we have observed at moderate concentrations of GdnHC1 could represent an intermediate in the folding process, as has been proposed in the general model for protein folding 17, in which collapse of secondary structural elements occurs prior to acquisition of the correct tertiary structure, or could represent the folding of independent structural domains. RfBP shares many characteristics in common with other egg-white proteins. It is rich in disulphide bridges (cf. ovotransferrin, ovomucoid, ovoinhibitor and lysozyme), it is glycosylated (cf. ovalbumin, ovotransferrin, ovomucoid, ovoinhibitor) and it is phosphorylated (cf. ovalbumin )1s. All these chemical modifications are the result of posttranslational modification. The detailed mechanisms of these post-translational mechanisms occurring in the oviduct have not been studied. In other eukaryote secretory cells it is generally accepted that as the nascent proteins are extruded through the rough endoplasmic reticulum they fold spontaneously~9. Glycosylation occurs as the proteins pass into the lumen; in the case of ovalbumin it has been shown to occur before the synthesis of the C-terminus is completed 2°. The disulphide isomerase which catalyses the isomerization of disulphides is loosely associated with the rough endoplasmic reticulum. The location of phosphorylation is not known. It therefore seems likely that the folding may occur in vivo, after the initial glycosylation, possibly before the formation of the final disulphide bridges, and probably before phosphorylation.

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It is interesting in the light of this to consider the mechanism of folding of the reduced RfBP. The reduced RfBP has a conformation intermediate between that of the native state and the fully denatured protein. Other proteins in which the disulphide bridges have been reduced fall into three categories. Small proteins, such as pancreatic trypsin inhibitor 21 and ribonuclease A 22 have c.d. spectra similar to those of the fully denatured state. Larger proteins, such as the F¢ fragment of immunoglobulin23 and human growth hormone 24 have c.d. spectra almost identical with those of the native proteins. The egg-white protein, ovotransferrin and a 36.4 kDa fragment of its N-terminus, on reduction of the disulphide bonds, show c.d. spectra consistent with a partially folded conformation25. The c.d. spectra varied with the temperature suggesting that the intermediate was less stable at 0°C than at 6°C. The intermediate could be oxidized by oxidized glutathione at 6°C to yield the renatured protein with the full iron-binding capacity restored. The properties of RfBP we have observed appear similar in this respect to those of ovotransferrin. The folding of RfBP in vivo may occur before it becomes phosphorylated, whereas our in vitro work on the refolding of reduced RfBP has studied the phosphorylated protein. Phosphorylation of RfBP appears to be important in targeting RfBP to the developing oocyte, since this is impaired in dephosphorylated R f B P 26. However, in view of the positions of the phosphorylation sites, clustered near the C-terminus, and presumably exposed on the surface of the fully folded protein, it seems unlikely that phosphorylation would have a major influence on the folding process. It would be of considerable interest to study the folding of deglycosylated and/or dephosphorylated RfBP to shed further light on folding in vivo.

Acknowledgements We thank the Science and Engineering Research Council for provision of the c.d. facility, and to Dr S. Provencher for supplying the CONTIN Program.

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

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