Stopped-flow analysis of the refolding of hen egg white riboflavin binding protein in its native and dephosphorylated forms

Biochimica et Biophysica Acta 1382 Ž1998. 157–166

Stopped-flow analysis of the refolding of hen egg white riboflavin binding protein in its native and dephosphorylated forms David A. McClelland 1, Nicholas C. Price

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Department of Biological and Molecular Sciences, UniÕersity of Stirling, Stirling FK9 4LA, UK Received 14 August 1997; revised 9 October 1997; accepted 9 October 1997

Abstract In earlier work ŽD.A. McClelland, S.H. McLaughlin, R.B. Freedman, and N.C. Price, Biochem. J. 311, 133–137 Ž1995.., we had shown that during the refolding of hen egg white riboflavin binding protein ŽRfBP. 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 Žf 8 ms.. 80% of the native protein fluorescence was regained within the dead-time of the instrument in fluorescence mode Ž1.7 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 ŽANS. 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. q 1998 Elsevier Science B.V. Keywords: Riboflavin binding protein; Refolding; ŽHen egg white.

1. Introduction Hen egg riboflavin binding protein Ž RfBP. acts as a source of riboflavin to the developing embryo and

Abbreviations: RfBP, riboflavin binding protein; GdnHCl, guanidinium chloride; ANS, 1-anilino-8-naphthalenesulphonate; CD, circular dichroism ) Corresponding author. Fax: 44 1786 464994 1 Present address: MRC Virology Unit, Church Street, Glasgow G11 5JR, UK.

possibly as an anti-microbial agent w1x. 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. The recent solution of the crystal structure of RfBP w2x has confirmed the suggestion w3x that this quenching is due to the stacking of aromatic groups within the hydrophobic binding pocket. The quenching provides a convenient assay for the integrity of the riboflavin-binding site of the protein.

0167-4838r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 7 - 4 8 3 8 Ž 9 7 . 0 0 1 7 9 - 9

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RfBP consists of a single polypeptide chain of 219 amino acids of molecular mass 29.2 kDa. The crystal structure w2x of the protein indicates that it has a novel fold, with a highly distinctive ligand-binding domain running from the N-terminus to about Cys 169. Two of the six a-helices have three residue interruptions which induce bends changing their direction. The areas of b-structure are very complex with a number of twists, bends and gaps. RfBP undergoes a number of post-translational modifications, namely: the formation of nine disulphide bonds w4x, extensive glycosylation on Asn 36 and Asn 147 w5x, and the phosphorylation of eight serine side chains from between Ser 186 and Ser 197 w2x. These phosphoserines, which are present in a highly anionic region of the molecule Ž186–199. which also includes five glutamate residues, are thought to be involved with serum RfBP uptake to the yolk. Together with the relatively small size of the protein, these features make RfBP an ideal system in which to study the effect of post-translational modifications on refolding. In previous work on the unfolding and refolding of RfBP, we demonstrated that, provided the disulphide bonds remained intact, the protein could be refolded from its denatured form with high efficiency w6x. In these experiments, manual mixing techniques were used to reduce the GdnHCl concentration from 6 to 0.1 M to initiate refolding. Essentially, all of the change in the far UV CD signal and f 90% of the change in protein fluorescence intensity occurred within the 10 s dead-time of the manual mixing. The remaining 10% of fluorescence change occurred over a further two hours. In the present paper, we report studies of the kinetics of the early stages of refolding in more detail, using stopped-flow rapid mixing techniques. We have also used the fluorescent probe 1-anilino-8naphthalenesulphonate ŽANS. to test for the possible involvement of the ‘‘molten globule’’ state in the refolding process w7,8x. Earlier work has shown that it is possible to remove the phosphoryl groups from mature RfBP using acid phosphatase w9x. We show that this treatment does not affect the ability of the protein to bind riboflavin, and has little detectable effect on the various kinetic phases of the refolding process.

2. Experimental section 2.1. Purification of RfBP Hen egg white RfBP was purified by the method of Hamazume et al. w4x as modified by Walker et al. w10x scaled up to process 400 ml of egg white. The eggs were obtained from local commercial sources. The holo-protein complex was isolated via ion-exchange chromatography, on two Sephadex A50 DEAE columns Ž10 = 9.6 cm2 . . The second column gives pure holo-RfBP, from SDS-PAGE studies. The apoprotein was obtained by ion-exchange chromatography of the holo-protein, on a SP Sephadex C50 colum n Ž 12 = 3.1 cm 2 . , at pH 3.8 Ž the riboflavinrRfBP complex dissociates between pH 3.8 and 4 w1x.. SDS-PAGE does not give an accurate estimation of the M r of RfBP due to its high degree of phosphorylation and glycosylation w1x. Protein and riboflavin concentrations were determined spectrophotometrically, as previously described w11x. The activity of the purified RfBP was verified by its ability to quench the fluorescence of riboflavin Žexcitation and emission wavelengths 370 and 520 nm, respectively.. The titration showed that complete quenching was observed at a 1:1 molar ratio of RfBP to riboflavin, indicating that the protein was fully active. The structural integrity of the RfBP was also assessed by CD and intrinsic protein fluorescence measurements. Manual mixing CD and fluorescence studies were performed on a JASCO J-600 spectropolarimeter and a Perkin-Elmer LS50B spectrofluorimeter, respectively as previously described w6x. 2.2. Dephosphorylation of RfBP Apo-RfBP was dephosphorylated essentially according to the method of Rhodes et al. w9x. 1.5 ml of the solution of RfBP Ž2 mgrml. was dialysed against 0.05 M sodium acetate at pH 5.3. The dialysis tubing was then placed in a universal tube filled with 30 ml of buffer, at 378C. 15 ml of potato acid phosphatase Ž0.9 units. ŽBoehringer Mannheim, grade 1. was added to the RfBP solution in the dialysis tubing, followed by an additional 5 ml Ž 0.3 units. after 3 h

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incubation. 1 ml samples of buffer Ž outside the dialysis bag. were removed from the universal tube at stated times Ž1–4 and 20 h. and analysed for inor-

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ganic phosphate, by a method adapted from Ames and Dubin w12x. 0.5 ml of 2% Žwrv. ammonium molybdate and 0.5 ml of 10% Žwrv. ascorbic acid

Fig. 1.

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8 cm2 , preswollen in buffer.. The column was washed with 0.05 M sodium acetate buffer Ž pH 7.0. , until the A 280 of the effluent was equal to that of the buffer Žthe acid phosphatase is not retained by the column.. The bound RfBP was eluted with this buffer containing 0.5 M NaCl. Fractions containing RfBP were pooled, dialysed against 0.05 M sodium acetate at pH 7.0 and freeze-dried. The freeze-dried material was resuspended in water to give a protein concentration of about 10 mgrml. Dephosphorylated and native RfBP were subjected to SDS-PAGE w13x. Half of the gel was stained for protein using Coomassie blue; the other half was stained for the presence of phosphate as described by Cutting w14x. 2.3. Refolding of denatured RfBP

Fig. 1. The refolding of RfBP monitored by changes in far UV CD and protein fluorescence. 2.5 mgrml Ž86 mM. RfBP was denatured by incubation in 50 mM sodium phosphate buffer ŽpH 7.0., containing 6 M GdnHCl at 208C for 15 min. Refolding was initiated by dilution with 10 volumes of buffer, to give a final concentration of GdnHCl of 0.55 M. ŽA. Changes in ellipticity at 230 nm during the refolding of apo-protein. Under these conditions, the ellipticity values at 230 nm of RfBP in the presence of 0.55 and 6 M GdnHCl were y68"6 and y19"5 mdeg, respectively. ŽB–E. Changes in fluorescence monitored at 350 nm with excitation at 290 nm ŽB and C represent apo-protein; D and E protein in the presence of equimolar riboflavin.. The traces shown are the averages of 20 shots with a filter of 2 ms ŽA, B and D., and 5 shots with a filter of 50 ms ŽC and E.. The solid lines shown in traces B–E are the fits to first order processes with the half times mentioned in the text. Under these conditions, the signals due to denatured RfBP were 1.6, 1.7, 2.2 and 2.2 units for traces B, C, D and E, respectively.

Žboth in H 2 O. were added to the 1 ml samples. A buffer blank was also prepared. The samples were mixed well, boiled for 5 min and left to cool for 30 min before the A 800 values were recorded. A standard phosphate curve was produced over the range 0–60 nmol phosphate. In order to remove the acid phosphatase from the RfBP, the contents of the dialysis sac were loaded onto a Whatman DE52 ion exchange column Ž 0.79 =

The protein was denatured by a 15 min incubation Žat 208C. in 50 mM sodium phosphate buffer ŽpH 7.0. containing 6 M GdnHCl, at a protein concentration of 2.5 mgrml. Concentrations of GdnHCl were checked by refractive index measurements w15x. Refolding was initiated by dilution of the sample with buffer to lower the GdnHCl concentration. Dilution was carried out by either manual or stopped-flow mixing for different experiments. Stopped-flow far UV CD studies were carried out at 208C on an Applied Photophysics SX-17MV Stopped-Flow Reaction Analyser instrument, using the umbilical cell in the 0.2 cm pathlength position. Unless otherwise stated, the mixing was in a 10:1 ratio Ži.e. 11-fold dilution. of protein in 6 M GdnHCl, so that the final concentration of GdnHCl present during refolding was 0.55 M. This concentration of denaturant has only very minor effects on the structure of the protein, and does not affect binding of riboflavin w11x. Studies of the refolding of RfBP measured the change in ellipticity at 230 nm against time; this wavelength was chosen as it gave better signal to noise than 225 nm. The dead-time of the instrument in the CD mode was determined by monitoring the refolding of lysozyme after denaturation in 6 M GdnHCl, since this reaction has been studied in considerable detail w16–18x. In agreement with these earlier studies, we observed two principal phases in the regain of ellipticity in the far UV, namely an overshoot in the early phase of the refolding followed

D.A. McClelland, N.C. Price r Biochimica et Biophysica Acta 1382 (1998) 157–166

by a slower decline of signal to that of the native protein. From the amplitude of the ellipticity change before the overshoot is complete, the dead-time of the instrument could be estimated as 8 ms. Fluorescence stopped-flow studies were recorded on the SX-17MV instrument, at 208C. Protein refolding was recorded by emission at 350 nm Ž with excitation at 290 nm., against time. Quenching of riboflavin fluorescence was recorded at 520 nm Ž with excitation at 370 nm., against time. All slit widths were typically 1 mm, but were adjusted to cater for different proteinrriboflavin concentrations. Unless otherwise stated, mixing was in a 10:1 ratio. The dead-time of the stopped-flow instrument in the fluorescence mode was determined using the reaction between 2,6-dichlorophenolindophenol and L-ascorbic acid w19x as being 1.7 ms. Kinetic analysis was performed using the ProrK software supplied with the instrument. In general, the half times for the various processes were estimated to have an error of "15%. To determine the start and end points of the refolding reactions, the ellipticity and fluorescence intensities of RfBP were measured after incubation in 6 M GdnHCl Žstart point. and 0.55 M GdnHCl Žend point. . Experiments concerning the regain of secondary and tertiary structure during refolding were duplicated in the presence of an equimolar concentration of riboflavin.

presence of an equimolar concentration of riboflavin had no detectable effect on the extent and rate of the changes in the far UV CD on refolding Ždata not shown.. 3.2. Changes in fluorescence of RfBP during refolding A total of four kinetic phases of fluorescence change during the refolding were resolved, namely: very fast; fast; slow; and very slow Ž Fig. 1 ŽB.,ŽC.. and Table 1.. Most Ž80%. of the total fluorescence change Ždefined as the difference between the fluorescence of RfBP in 6 M GdnHCl and in 0.55 M GdnHCl. occurred in the dead-time of the instrument, estimated to be 1.7 ms. The first order rate constants of the second and third phases Ž fast and slow. were determined by the ProrK kinetic analysis software supplied with the instrument. The half time of the very slow phase was estimated from the results of manual mixing refolding experiments of the type described previously w6x ŽTable 1.. When refolding of RfBP was performed in the presence of riboflavin, four kinetic phases were also

Table 1 Properties of intermediates in the refolding of hen egg white RfBP

2.4. ANS as a structural probe during refolding Denatured RfBP was refolded in the presence of 20 mM ANS, and the fluorescence of ANS measured Žexcitation 370 nm, emission 480 nm. . The interactions between ANS and native RfBP were also investigated by measurements of ANS fluorescence and by studies of riboflavin binding.

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Intermediate U I1 Property Far UV CD Fluorescence ANS binding Riboflavin binding

0 0 0 0

100 1 80 100 0

I2

I3

N

100 90 100 100

100 95 100 100

100 100 100 100

In the table, the intermediates refer to those in the scheme: 1

2

3

4

U™I 1™I 2™I 3™N.

3. Results 3.1. Regain of secondary structure of RfBP during refolding The regain of secondary structure, as measured by the regain of CD signal at 230 nm was found to be complete within the dead-time of the stopped-flow instrument Žestimated to be 8 ms. Ž Fig. 1ŽA... The

The half times for steps 1, 2, 3 and 4 are -1.7 ms, 30 ms, 46 s and 60 min for the apo-protein; however, the half time for the second step is approximately doubled in the presence of riboflavin. The half times for the steps in the case of dephosphorylated protein are -1.7 ms, 25 ms, 50 s and 60 min, respectively. The values shown represent the % of the property of the native protein. 1 Since the dead-time for the stopped-flow CD is )1.7 ms, it is not certain that I 1 has regained all the native far UV CD signal; however, the change is complete by 8 ms, i.e. well before I 2 is formed.

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observed Ž Fig. 1 ŽŽD., ŽE.. , indicating that the mechanism of folding was similar in the presence of the ligand. The half time of the second Ž fast. phase was increased Župto 60 ms. ; the half times of the others were not significantly different from those of the apo-protein. It should be noted that the amplitude of the total fluorescence change is some 80% greater than in the case of the apo-protein, since the protein fluorescence is markedly quenched by bound riboflavin w1x. 3.3. Regain of riboflaÕin binding actiÕity during refolding

strument Ž1.7 ms.. These results indicate that the correct binding site for riboflavin is formed during the second phase of tertiary structural changes in the protein, and that this is followed by a rapid association of the folded protein with riboflavin. When equimolar concentrations Ž7.8 mM. of native and apo-RfBP were mixed at a GdnHCl concentration of 0.55 M, the quenching of riboflavin fluorescence occurred with a t 1r2 of 2.5 ms, corresponding to a second order rate constant of 5.2 = 10 7rMrs. This value is similar to the values of rate constants for association of enzymes and substrates w20x. 3.4. Effect of ANS on natiÕe and refolding RfBP

The regain of riboflavin binding activity was measured through the quenching of riboflavin fluorescence at 520 nm when riboflavin was included in equimolar amounts during the refolding of RfBP. The rate of fluorescence quenching was found to have similar kinetics to the second Ž fast. phase observed in changes in protein fluorescence Žwhen measured in the presence of riboflavin., with a t 1r2 of 65 ms ŽFig. 2.. There was no change in the riboflavin fluorescence during the dead-time of the stopped-flow in-

The addition of 20 mM ANS to apo-RfBP Ž8 mM., resulted in a 20-fold increase in ANS fluorescence at 480 nm. The intrinsic fluorescence of RfBP was quenched by f 50%, and the l max slightly blue shifted, from 346 to 343 nm Ž Fig. 3 Ž A.. . At least part of the quenching of the protein fluorescence may be due to energy transfer from protein to ANS. An equivalent quenching of the protein fluorescence and blue shift was found when the experiment was re-

Fig. 2. Changes in the fluorescence of riboflavin during the refolding of RfBP. Riboflavin fluorescence was monitored at 520 nm, with excitation at 370 nm. The traces represent the fluorescence of ŽA. riboflavin mixed with buffer, ŽB. riboflavin mixed with RfBP in 0.55 M GdnHCl, and ŽC. riboflavin mixed with denatured RfBP, so that refolding occurs. In each case, the final riboflavin concentration was 7.8 mM; in B and C, the final concentration of RfBP was 7.8 mM. The theoretical curve for trace B is for a second order process with k s 5.2 = 10 7rMrs; that shown for trace C is for a first order process with k s 10.5rs.

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peated with the holo-protein Ždata not shown. ; in this case, the enhancement in ANS was identical to that found with the apo-protein. It should be noted that some other proteins, e.g. glutamate dehydrogenase

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w21x and creatine kinase w22x can also bind ANS in their native states. Addition of ANS did not affect the ability of RfBP to bind riboflavin Ž data not shown. . By contrast, denatured RfBP Ž in 6 M GdnHCl. was unable to bind ANS, as there was no enhancement of ANS fluorescence in this case. During the refolding of denatured RfBP, the ability to bind ANS was regained within the dead-time of the instrument Ž1.7 ms. , i.e. before the regain of riboflavin binding ability Ždata not shown.. The presence of 20 mM ANS during refolding did not affect the ability of the refolded protein to bind riboflavin, nor did it affect the kinetics of protein fluorescence or riboflavin fluorescence changes ŽFig. 3 ŽB.. . 3.5. Dephosphorylation of RfBP The phosphate released from RfBP after treatment with the acid phosphatase corresponded to 8.2 " 0.2 molrmol protein, indicating that all eight phosphate groups known to be present in the mature protein w5x had been removed. The treated protein ran f 5% faster than the native protein on SDS-PAGE and, in contrast to the native protein, did not stain for the presence of phosphate. In terms of the far UV CD spectrum Ž Fig. 4., the overall structure of the dephosphorylated protein was similar to that of native protein; this was confirmed by the near identity of the fluorescence spectra of the two proteins and the similar pattern of riboflavin binding. The unfolding of dephosphorylated RfBP by GdnHCl occurred in a similar biphasic fashion to native RfBP with a plateau region between 3 and 5 M GdnHCl w11x. Addition of ANS to the dephosphorylated protein resulted in a somewhat higher Ž10%. ANS fluorescence increase; this could reflect enhanced binding of the negatively

Fig. 3. Effect of ANS on RfBP. ŽA. Effect of ANS Ž20 mM. on the fluorescence of apo-RfBP Ž8 mM.. Excitation was at 290 nm; solid and dashed lines refer to the absence and presence of ANS, respectively. ŽB. Effect of ANS Ž20 mM. on the refolding of RfBP monitored by the changes in riboflavin fluorescence. Traces Ži. and Žii. refer to the absence and presence of ANS, respectively. The difference between the traces reflects the small contribution made by ANS to the total fluorescence in Žii.. Conditions were analogous to those used in Fig. 1 and Fig. 2. There was no change in the riboflavin fluorescence within the dead-time of the stopped-flow apparatus.

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Fig. 4. Far UV CD spectra of native and dephosphorylated RfBP. The solid and dashed lines represent native and dephosphorylated apo-RfBP, respectively.

charged ANS to a protein from which considerable negative charge had been removed. The refolding of dephosphorylated RfBP after denaturation was very similar to that of native protein, using either manual mixing or stopped-flow mixing techniques, with four kinetic phases in fluorescence changes being resolved, and similar kinetics of regain of riboflavin binding. The half times for the kinetic phases are shown in Table 1. Taken together these data indicate that the removal of the eight phosphate groups from RfBP has little effect on the structure, stability, activity or refolding of the protein.

4. Discussion As long as the nine disulphide bonds remain intact, denatured RfBP was found to refold completely and very quickly after the removal of the denaturing agent, without the need for any other additional agents such as chaperone proteins. From the stopped-flow and steady-state data, it is apparent that the refolding process occurs in several stages. The

results show that all of the native secondary structure and a large proportion of the native tertiary structure are formed very quickly after the initiation of refolding, within the dead-times of the respective instrument modes Ž 8 and 1.7 ms, respectively. . This rapid formation of both of native secondary structure and a relatively compact intermediate has been observed during the refolding of several other proteins e.g. lysozyme, cytochrome c, a-lactalbumin and apomyoglobin w16,23–25x and led to the development of the concept of ‘‘molten globule’’ intermediates in folding pathways w8x. Additional evidence of the participation of ‘‘molten globule’’ intermediates has been gained from the use of the fluorescent probe ANS, which for many proteins binds only weakly to the native and fully unfolded forms of proteins, but strongly to the ‘‘molten globule’’ state w7,26,27x. However, it should be noted that recent studies have shown that, at least for some proteins, the presence of ANS from the start of refolding can perturb the folding process, leading to the formation of ANS-dependent kinetics and intermediate states w28x. In the case of RfBP, it is clear that the presence of ANS has little effect on the kinetics of the refolding process. However, this technique is unable to provide any evidence for the possible involvement of a ‘‘molten globule’’ type intermediate in the refolding of RfBP, since the native protein binds ANS to a significant extent. Nevertheless, the results obtained in the presence of ANS do yield some additional insights into the refolding process. During refolding, the protein regained the ability to bind ANS more quickly than the ability to bind riboflavin. Since the holo-protein can bind ANS, in a similar manner to the apo-protein, it is clear that the ANS is binding at a site, or sites, distinct from the riboflavin binding pocket. These sites, which presumably involve clusters of hydrophobic residues, are formed within the dead-time of the stopped-flow instrument, i.e. in the first Žvery fast. phase in which the secondary structure is formed and in which the majority Ž 80%. of the total change in protein fluorescence occurs. The formation of the more highly structured hydrophobic pocket to which riboflavin binds tightly occurs at a later stage, corresponding to the second Žfast. phase of the protein fluorescence change Ž the t 1r2 of this step is 30 ms in the apo-protein, but is approximately doubled in the presence of riboflavin..

D.A. McClelland, N.C. Price r Biochimica et Biophysica Acta 1382 (1998) 157–166

This second phase presumably involves a further change in the tertiary structure of the protein, resulting in formation of near native structure. The effect of riboflavin in slowing the rate of this step presumably arises from constraints placed by the ligand on small adjustments in the local tertiary structure. This situation has some analogies to that described by Mucke and Schmid w29x, who found that under certain conditions the presence of intact disulphide bonds decelerated the folding of ribonuclease T1, presumably by restricting local structural rearrangements. The third Žslow. phase Ž t 1r2 s 46 s. of the fluorescence change in RfBP presumably reflects further small changes in the tertiary structure. The final regain of native tertiary structure can take up to 2 h. This Žvery slow. phase could reflect the cisrtrans isomerisation of X-Pro bonds w30,31x, although detailed studies with added PPI and further analysis of the recent crystal structure of RfBP w2x would be necessary to confirm this proposal. It is to be noted that the adjustments of tertiary structure in the fast, slow and very slow phases do not lead to any enhancement in the binding of ANS. RfBP was readily dephosphorylated by the potato acid phosphatase. From the fluorescence and binding analysis, however, it is clear that no gross overall structural change occurs upon dephosphorylation, and that the riboflavin-binding site is conserved. The kinetics of the refolding reaction for dephosphorylated RfBP, in the presence and absence of riboflavin, were found to be identical to those of the native protein as were the kinetics of regain of riboflavin binding activity by refolding and the native protein. It is thus, clear that the presence of the cluster of eight negatively charged phosphate groups has little effect on the pathway of refolding; and indicates that these residues are not located close to any nucleating centre of the molecule. Taken together with the accessibility of the phosphate groups to the action of the phosphatase, this suggests that in vivo phosphorylation occurs only after folding is fully or very largely completed, presumably at residues which form part of a surface loop of the protein. It is interesting to note that in the crystal structure of the protein w2x, the phosphorylated region is not ordered but that the polypeptide chain immediately before and after it folds clearly into the last two of the six a-helices of the molecule.

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Acknowledgements We wish to thank the University of Stirling for provision of a studentship Žto D.A.M.. , the Biotechnology and Biological Sciences Research Council for provision of the CD and stopped-flow facilities and Dr. Sharon Kelly for helpful discussions.

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