α-Crystallin and ATP facilitate the in vitro renaturation of xylanase: enhancement of refolding by metal ions

Abstract

α-Crystallin is a multimeric protein that functions as a molecular chaperone and shares extensive structural homology to small heat shock proteins. For the functional in vitro analysis of α-crystallin, the xylanase Xyl II from alkalophilic thermophilic Bacillus was used as a model system. The mechanism of chaperone action of α-crystallin is less investigated. Here we studied the refolding of Gdn HCl-denatured Xyl II in the presence and absence of α-crystallin to elucidate the molecular mechanism of chaperone-mediated in vitro folding. Our results, based on intrinsic tryptophan fluorescence and hydrophobic fluorophore 8-anilino-1-naphthalene sulfonate binding studies, suggest that α-crystallin formed a complex with a putative molten globule-like intermediate in the refolding pathway of Xyl II. The α-crystallin•Xyl II complex exhibited no functional activity. Addition of ATP to the complex initiated the renaturation of Xyl II with 30%–35% recovery of activity. The nonhydrolyzable analog 5′-adenylyl imidodiphosphate (AMP-PNP) was capable of reconstitution of active Xyl II to a lesser extent than ATP. Although the presence of Ca²⁺ was not required for the in vitro refolding of Xyl II, the renaturation yield was enhanced in its presence. Experimental evidence indicated that the binding of ATP to the α-crystallin•Xyl II complex brought about conformational changes in α-crystallin facilitating the dissociation of xylanase molecules. This is the first report of the enhancement of α-crystallin chaperone functions by metal ions.

It is well established that the amino acid sequence of a polypeptide chain contains the information that determines the three-dimensional structure of a functional protein (Anfinsen 1973). However, the folding of many proteins in vivo requires the assistance of a preexisting machinery of molecular chaperone proteins (Ellis 1987; Creighton 1990). Chaperones are catalysts in the sense that they transiently interact with their substrate proteins but are not present in the final folded product, and in that they increase the yield of folded protein. Despite the fact that in vitro folding may not exactly mimic the intracellular environment in the cell, it is minimally a good model for in vivo protein folding (Fink 1999). In the past decade, wide interest has been generated in crystallins, a group of structural proteins initially thought to be lens-specific, which have also been identified in nonlenticular tissues (Klemenz et al. 1991; Iwaki et al. 1992). α-Crystallin shares both sequence and structural homology with small heat shock proteins (sHSPs) and behaves in many ways like them (Ingolia and Craig 1982; Klemenz et al. 1991; De Jong et al. 1993). The chaperone-like activity of α-crystallin may be important in the formation and maintenance of eye lens transparency, and the age-related deterioration of the chaperone function may contribute to the development of cataracts (Horowitz 1993). The expression of α-crystallin has been shown to be induced by thermal (Klemenz et al. 1991) and hypertonic stress (Kelley et al. 1993). α-Crystallin has been reported to be functionally equivalent to the sHSPs murine Hsp25 and human Hsp27 in the in vitro refolding of α-glucosidase and citrate synthase, respectively (Jakob et al. 1993); it was unable to refold rhodanase denatured in 6 M Gdn HCl, however (Das et al. 1996). α-Crystallin has also been reported to bind the temperature-induced molten globule state of proteins (Rajaraman et al. 1996) and prevent photoaggregation of γ-crystallin by providing hydrophobic surfaces (Raman and Rao 1994).

Several studies have supported a functional relationship between ATP and sHSPs, and a role of ATP in the refolding of proteins by sHSPs has been described (Liberek et al. 1991). Even though equilibrium binding studies, intrinsic tryptophan fluorescence, and ³¹P nuclear magnetic resonance spectroscopy have demonstrated an interaction between ATP and bovine α-crystallin (Reddy et al. 1992), the role of ATP in the mechanism of folding of proteins by α-crystallin is not well understood. ATP is an abundant phosphorous metabolite present in high concentrations in lens cells from many species. High concentrations of ATP are present in the skeletal muscle (Burt et al. 1976), in which high levels of α-crystallin are coexpressed (Bennardini et al. 1992). Despite the growing interest in the chaperone action of α-crystallin, little is known about its mechanism of chaperoning. For the functional in vitro analysis of α-crystallin, the xylanase Xyl II from alkalophilic thermophilic Bacillus has been used as the model system. Xylanases have raised enormous interest in the past decade in view of their application in the clarification of juices and wines, conversion of renewable biomass into liquid fuels, and in the development of environmentally sound biological or prebleaching processes in the paper and pulp industries (Kulkarni et al. 1999). Despite the tremendous biotechnological potential of xylanases, there are comparatively fewer reports of unfolding/folding studies of this class of enzymes and especially of enzymes from extremophiles. We have reported structure-function studies elucidating the contribution of essential amino acids to the catalytic mechanism of Xyl II (Dey et al. 1992; Nath and Rao 1998). In the present study, experimental findings indicate that α-crystallin forms a complex with a putative molten globule-like intermediate in the refolding pathway of Xyl II. ATP-dependent release of Xyl II, and enhancement of the reactivation yield were investigated. We focused our attention on the effect of metal ions in the α-crystallin-mediated refolding of Xyl II and showed for the first time the enhancement of the refolding yield of the enzyme in their presence. Our present investigations of α-crystallin-assisted refolding in the presence of ATP and metal ions may shed light on the mechanism of chaperoning in vivo.

Results and Discussion

In vitro refolding of Xyl II

The renaturation of Xyl II was initiated by diluting the enzyme treated with 6 M guanidine hydrochloride in buffer at 37°C. The time-dependent regain of activity at different enzyme concentrations exhibited a clear dependence on the initial protein concentration. The reactivation yield appeared to reach a maximum at 10 μM Xyl II concentration. The spontaneous Xyl II renaturation experiments were performed in parallel with the α-crystallin-assisted renaturation experiments. Significantly, under the conditions described, the enzymatic activity of native Xyl II was not influenced by the presence of even the largest concentration (0.8 mg/mL) of α-crystallin tested (Table 1).

Table 1.

Effect of α-crystalline on xyl II activity

α-crystalline (μg)	Activity (IU/ml)
0.1	6.00
0.3	5.95
0.5	5.90
0.8	6.00

Xyl II (10 μM) was incubated with different concentrations of crystalline for 2 h at 37°C, and enzyme activity was checked as described, in IU/ml.

Intrinsic tryptophan fluorescence spectroscopy (ITF)

The intrinsic fluorescence of α-crystallin-bound Xyl II as a measure of tertiary structure was examined. The native enzyme has a fluorescence wavelength maximum at 339 nm, which changed markedly in the unfolded state with a shift in the emission maximum to 353 nm (λ_ex 295 nm). Generally, increased exposure of tryptophan (Trp) residues to solvent on unfolding results in a red shift of the Trp fluorescence. The wavelength of the maximum emission of α-crystallin-bound Xyl II was at 343 nm, which indicated that the Trp residues were still in a nonnative, but in a more hydrophobic environment than in the fully unfolded state (Fig. 1 ▶).

Fig. 1.

Conformational properties of chaperone-associated xylanase. Xyl II at a concentration of 100 μM was incubated with 6 M Gdn HCl for 16 h, and further diluted in 50 mM phosphate buffer, pH 7.2, containing 0.2 mg/mL α-crystallin at 37°C. After incubation for 30 min, the tryptophan fluorescence was recorded. Trp fluorescence of native Xyl II (trace a), α-crystallin-bound Xyl II (trace b), denatured Xyl II (trace c), and α-crystallin (trace d). All samples were excited at 295 nm.

ANS binding

Attempts were made to determine the structural characteristics of Xyl II bound to α-crystallin by using a fluorescent probe, 1-anilino-naphthalene-8-sulphonate (ANS). ANS was not fluorescent in aqueous solutions (λ_em 525 nm); however, on addition of proteins containing hydrophobic pockets, its emission maximum shifted to shorter wavelengths, and the emission intensity was enhanced. Xyl II bound by α-crystallin showed significant increase in ANS fluorescence, which was blue shifted to 490 nm, indicating exposure of hydrophobic surfaces in the folding intermediate of Xyl II (Fig. 2 ▶). In contrast, the fluorescence of native as well as the denatured enzyme showed no binding to ANS. This intermediate was probably similar to the "molten globule" states proposed as an accessible conformation for several proteins (Dolgikh et al. 1982).

Fig. 2.

Fluorescence spectra of ANS bound to (▪) α-crystallin•Xyl II complex; (♦) native Xyl II; (▴) Gdn HCl-denatured Xyl II; (▾) refolded enzyme; and (○) α-crystallin. Forty μM ANS was added to each sample and the fluorescence was recorded at 490 nm. The samples were excited at 375 nm.

Addition of ATP to the α-crystallin•Xyl II complex initiates renaturation

The addition of ATP to the refolded enzyme in the presence of α-crystallin•Xyl II complex initiated the renaturation of Xyl II (Fig. 3 ▶). The refolded enzyme was probably in a reactivatable form, because after 1 h, when ATP was added, the recovery of enzyme activity (30%–32%) was obtained. The peak fluorescence intensity at 340 nm showed a decrease of 26% in the presence of 0.3 mM ATP, compared to that of the complex observed in the absence of ATP (Fig. 4 ▶). Fluorescence spectra of native Xyl II in the absence or presence of ATP did not show any alteration (Fig. 4 ▶), indicating that the change in conformation may be due to the interaction of ATP with α-crystallin in the complex.

Fig. 3.

The refolding of Xyl II in the presence of various factors as monitored by enzymatic activity. The molar concentration of Xyl II was 1 μM. Refolding in presence of (▴) buffer alone; (▪) 1 mg/mL of BSA or (•) or PEG; α-crystallin at varying concentrations (※) −0.1 mg/mL; (♦) −0.15 mg/mL; (▾) −0.2 mg/mL; (×) −0.3 mg/mL. ATP was added to the refolding mixture to a concentration of 0.3 mM at the time indicated by an arrow.

Fig. 4.

Fluorescence spectroscopy of α-crystallin•Xyl II complex in the presence and absence of ATP. Shown are emission spectra for α-crystallin•Xyl II complex, in the absence of ATP (▪), in the presence of AMP-PNP (♦), and in the presence of ATP (•). ▴ and represent spectra of native Xyl II in the absence and presence of 0.3 mM ATP. Spectra of the buffers (including ATP) were subtracted from the spectra of the protein samples.

The α-crystallin-mediated renaturation of Xyl II was examined as a function of the chaperone concentration. As shown in Figure 5 ▶, 15% of the original Xyl II activity was recovered at the lowest concentration of α-crystallin (0.05 mg/mL). The extent of renaturation increased in a concentration-dependent manner, and a maximum of 30%–35% of the original activity was recovered at the α-crystallin concentration of 0.2–0.5 mg/mL. The increase in the α-crystallin concentration may have increased the collisional frequency so as to favor the formation of the complex. The ability of refolding was quite specific for α-crystallin, because bovine serum albumin (BSA) and PEG each had no effect on the refolding of Xyl II.

Fig. 5.

Reactivation of Xyl II at varying concentrations of α-crystallin. Xyl II was renatured as described in the legend to Figure 2 ▶ in the presence of varying concentrations of α-crystallin. After 1 h, ATP (0.3 mM) was added to each sample and incubated further. Aliquots were withdrawn after 4 h and assayed for Xyl II activity.

When unfolded Xyl II was allowed to renature in the absence of α-crystallin for 2 h and if α-crystallin and ATP were added at this time, there was no effect on the rate or extent of the observed renaturation (data not shown). This indicated that α-crystallin was not able to rescue misfolded Xyl II aggregates once they had formed.

Temperature dependence of the release of Xyl II from the complex on addition of ATP

The results of the temperature shift experiments of α-crystallin-assisted refolding of Xyl II are shown in Figure 6 ▶. In all cases, the complex had been previously formed by diluting unfolded Xyl II into a buffer containing α-crystallin at 37°C. The addition of ATP to the complex enabled the recovery of activity in the range 28° to 45°C, whereas there was no activity when the complex was shifted to 10°C. Thus, the temperature dependence of release of the active Xyl II may be due to the conformational changes that occurred at these temperatures. Equilibrium binding studies of ATP/α-crystallin conducted at 37°, 22°, and 4°C revealed temperature dependence of the interaction of ATP and α-crystallin. Binding occurred at 37°C but was not significant at 22°C and was absent at 4°C (Palmisano et al. 1995). Thus, the differential binding of ATP to α-crystallin was due to the observed modifications of α-crystallin between 25° and 45°C and may result in the temperature-dependent release of the active Xyl II.

Fig. 6.

Temperature-dependent refolding of Xyl II. Renaturation of Xyl II (100 μM) was initiated at 37°C by diluting 10 μL of the sample into a final volume of 1 mL activation of Xyl II (1 μM) in the presence of α-crystallin 0.2 mg/mL. After incubation for 1 h, the mixtures were supplemented with ATP (♦) and without ATP (▴) and further incubated at the indicated temperatures. Xylanase activity was then determined after incubation for 30 min prior to assay.

The effects of adenine nucleotides upon the α-crystallin-mediated reconstitution of active Xyl II were investigated. As shown in Figure 7 ▶, the addition of AMP-PNP, an ATP analog with a nonhydrolyzable β-γ bond, resulted in a maximum of 20% of Xyl II activity in 5 h. Addition of ADP and AMP did not have any effect on refolding.

Fig. 7.

Effect of adenine nucleotides on the α-crystallin-mediated reactivation of Xyl II. Renaturation of Xyl II (100 μM) was initiated at 37°C by diluting 10 μL of the sample into a final volume of 1 mL in the presence of α-crystallin 0.2 mg/mL. After 1 h the following additions were made: no nucleotide (♦), AMP (○), ADP (•), AMP-PNP (×) and ATP (▪). At indicated time intervals, 50-μL aliquots of the refolding solution were withdrawn and assayed for xylanase activity as described in Materials and Methods.

The ability of refolding was quite specific for α-crystallin, because BSA and PEG had virtually no effect on the refolding (Fig. 3 ▶). BSA and PEG facilitated reactivation of several proteins in an unspecific way (Cleland and Wang 1990). However, BSA as well as PEG did not exert a significant effect on the reconstitution of Xyl II.

Effect of metal on the renaturation profile

Experiments examining the effect of Ca²⁺ on α-crystallin-assisted Xyl II renaturation were performed in parallel with those where only α-crystallin and ATP were used. When Ca²⁺ was initially present with the preformed complex, the ATP-induced release of Xyl II was characterized by a substantial increase in the renaturation yield compared with the values observed with α-crystallin alone. When Ca²⁺ was added to the α-crystallin • Xyl II complex formed prior to the addition of ATP, the yield of the renatured enzyme was significantly higher (40%) than in the absence of the metal ion. The enhancement of the renaturation yield suggested that interaction of Ca²⁺ with the α-crystallin • Xyl II complex induced a conformational change in α-crystallin that permits final folding of Xyl II and weakened hydrophobic interactions so that Xyl II could be released. It was documented that the presence of calcium ions in lens water (Spector et al. 1974; Rink et al. 1977) may play a significant role in the stabilization of α-crystallin in vivo. It was also observed that changes in the order of addition of unfolded Xyl II and Ca²⁺ to α-crystallin resulted in significant differences in the renaturation yield. When α-crystallin was preincubated with Ca²⁺ prior to the addition of Xyl II and ATP, the extent of regain in activity was found to decrease (Fig. 8 ▶). We monitored the effect of addition of calcium ions to α-crystallin at 37°C (Fig. 9 ▶). The decrease in fluorescence intensity was attributed to a change in the environment of the tryptophan residues, causing minor conformational changes in α-crystallin. This was in contrast to our earlier results where the enhancement of refolding yield occurred in the presence of Ca²⁺. The change in the order of addition of Ca²⁺ played a significant part in the renaturation yield. This finding may be interpreted as follows: When Ca²⁺ was directly added to α-crystallin prior to the addition of denatured Xyl II, some minor conformational changes were observed in α-crystallin which may be related to a change in the extent of binding surfaces or its access, thereby preventing the formation of the complex. However, our results showed that metal ions alone did not effect the renaturation of Xyl II (data not shown), and thus the metal effect was secondary to the chaperone function. As it has been reported that no ion-complexing regions were detected in the primary structure of α-crystallin, it is likely that the Ca²⁺-induced aggregation of α-crystallin involved nonspecific binding (Jedziniak et al. 1972; Spector and Rothschild 1973; Spector et al. 1974). A similar increase in the renaturation yield was obtained when Mg²⁺ was used along with α-crystallin and ATP. Although the ionic radii of Ca²⁺ and Mg²⁺ are different, they seem to be effective in the refolding. Xylan showed a marginal increase in reactivation, which may be due to the substrate-induced protection or stabilization of the refolded enzyme.

Fig. 8.

Effect of metal ions on the α-crystallin-mediated reactivation of Xyl II. Renaturation of Xyl II was initiated by diluting 10 μL of the sample into 1 mL final volume of potassium phosphate buffer, pH 7.2 with α-crystallin (0.2 mg/mL). (a) Refolding in buffer alone, (b) refolding in the presence of α-crystallin and ATP, (c) Ca²⁺ (5mM), (d) Mg²⁺ (5 mM) was added to the preformed α-crystallin•Xyl II complex. (e and f) Ca²⁺ and Mg²⁺ 5 mM each were preincubated with α-crystallin prior to the addition of Xyl II.

Fig. 9.

Corrected fluorescence spectrum of α-crystallin at 37°C in 50 mM phosphate buffer, pH 7.2, in the absence (A) and presence (B) of 5 mM CaCl₂. Excitation was at 295 nm and the spectral bandpass values were 5 nm.

Our results demonstrated that α-crystallin behaved as a molecular chaperone by actively participating in the refolding and reactivation of Xyl II. The conditions for the unfolding of native Xyl II were sought in belief that the unfolded enzyme or its folding intermediates would serve as a substrate for the α-crystallin-mediated reconstitution of active Xyl II. We have shown that under ATP-depleted conditions, Xyl II is bound to α-crystallin. The presence of ATP was necessary for the recovery of the active enzyme or for the dissociation of Xyl II from α-crystallin. Conformational changes have been proposed to play a major role in the binding of folding intermediates and in the discharge of polypeptides from molecular chaperones. One of the signals for inducing such structural changes was the hydrolysis of ATP, as reported in the cases of the chaperones GroEL (Liberek et al. 1991) and DnaK (Mendoza et al. 1991). However, in some cases the chaperones GroEL (Gloubinoff et al. 1989) and BiP (Kassenbrock and Kelly 1989) did not require ATP hydrolysis. The mere binding of the adenine nucleotide to the chaperone induced a typological change in the chaperone but weakened its interaction with the bound protein. Studies performed on the refolding of lactate dehydrogenase (Badcoe et al. 1991) and dihydrofolate reductase (Viitanen et al. 1990) in the presence of GroEL indicated that the hydrolysis of ATP is not absolutely required for the dissociation of those enzymes from GroEL. They were released from GroEL in the presence of a nonhydrolyzable ATP analog, AMP-PNP. When similar experiments were performed on Xyl II, it was found that Xyl II dissociated from α-crystallin in the presence of ATP and to some extent in the presence of AMP-PNP. ADP and AMP did not facilitate the release of the enzyme. These results probably indicate that simple binding of ATP or an equivalent structure was sufficient to disrupt the complex between α-crystallin and Xyl II. The quenching of intrinsic fluorescence in the presence of ATP has been reported for both α-crystallin and for αB-crystallin, suggesting ATP-induced conformational changes (Palmisano et al. 1995; Muchowski et al. 1999). The role of ATP in the chaperone α-crystallin-mediated refolding has been less investigated. Earlier experiments in our laboratory on xylose reductase (Rawat and Rao 1998) revealed an enhancement in the refolding yield in the presence of α-crystallin and ATP. The refolding of citrate synthase by α-crystallin was enhanced twofold in the presence of ATP (Muchowski and Clark 1998).

The structural characteristics of Xyl II bound to α-crystallin were determined using a fluorescent probe, ANS. We could detect an intermediate state with enhanced ANS fluorescence at 490 nm. This suggests that α-crystallin probably bound to a putative molten globule state. Studies have shown that α-crystallin binds to molten globule states of xylose reductase (Rawat and Rao 1998) and carbonic anhydrase (Rajaraman et al. 1996). Our results do not completely rule out the possibility that Xyl II was undergoing refolding while bound to the surface of α-crystallin, when the protein-protein interaction involved in the binding is considered. It seems more plausible that the folding of Xyl II was initiated during the bound state, and the functional activity was obtained during the free state of equilibrium.

α-Crystallin has been reported to be induced by thermal or hypertonic stress (Dasgupta et al. 1992; Kelley et al. 1993), and its expression is markedly increased in a number of neurological disorders such as Creutzfeld-Jacob disease, Alexander disease, and Lewy body disease (Duguid et al. 1988; Iwaki et al. 1989; Iwaki et al. 1992). To decipher the mechanism of chaperone function, it was necessary to study the conformational aspects of target proteins, which are recognized by α-crystallin. The specificity of α-crystallin appears to be limited to specific conformational intermediates that occur on the denaturation pathway, with no affinity for the intermediates formed on the refolding pathway (Das and Surewicz 1995). The experimental evidence in the present study revealed that α-crystallin formed a complex with Xyl II in the refolding pathway. Similarly in the case of lysozyme, it was observed that α-crystallin inhibits the aggregation as well as the oxidative renaturation of lysozyme in the refolding pathway (Raman et al. 1997).

In the molecular mechanism of chaperone-mediated refolding of α-crystallin, the role of calcium ions has been less investigated. Calcium ions play a pivotal role in the physiology of cataract formation, which has been a topic of frequent investigation. The aqueous humor calcium ion concentration in a human eye with a normal transparent lens ranges from 0.5 to 2.0 mM, whereas it is far larger (0.1–64 mM) in a subject with cataractous lenses (Chattopadhyay et al. 1997). In addition, several physiological and biochemical processes in the eye lens are dependent on the Ca²⁺ ion concentration. Our present results show that Ca²⁺ is probably required to ensure the proper release and association of Xyl II in vitro, and its presence affected the observed renaturation yields. Ca²⁺ did not stabilize native Xyl II or help in the spontaneous refolding of the denatured enzyme. However, an investigation of the role of calcium in the protection of thermal unfolding of mesophilic xylanase from Pseudomonas fluorescens showed that occupancy of the calcium-binding domain with its ligand protected the enzyme from inactivation, whereas the addition of calcium or EDTA did not influence the catalytic activity of the xylanase (Spurway et al. 1997). The role of Mg²⁺ in the physiology of cataract is thus far unknown, and the enhancement of the refolding yield in the presence of Mg²⁺ merits further investigation. To our knowledge, the present study is the first demonstration of the α-crystallin-facilitated refolding of xylanase. The in vitro conditions which were used in the present study for the α-crystallin-mediated system may not represent the complex in vivo cellular conditions that are probably essential for extremophiles. The modulation of refolding of induced enzyme systems such as xylanase may require other chaperone and nonchaperone proteins. The translocation of a partially unfolded structure could be greatly facilitated compared to the native state. The folding of the protein might occur on the external side of the cytoplasmic membrane, because the metal binding properties of the cell wall possibly provide a high metal ion environment (Petit-Glatron et al. 1993). Investigation of α-crystallin-assisted renaturation of extremophilic xylanase in the presence of ATP and metal ions may shed light on the mechanism of chaperoning in vivo.

Materials and methods

ATP, ADP, AMP-PNP, and α-crystallin were purchased from Sigma. All chemicals used were of analytical grade.

Xylanase purification and assay

Purification of Xyl II from alkalophilic thermophilic Bacillus was as described (Nath and Rao 1998). Xylanase activity was assayed by mixing a 0.5-mL aliquot of enzyme in 50mM potassium phosphate buffer, pH 7.0 with 0.5 mL of 1% xylan at 50°C for 30 min. The reducing sugar released was determined by Miller’s method (Miller 1959) using D-xylose as a standard. One unit of xylanase activity was defined as the amount of enzyme that produced one μmole of xylose equivalent per min under the assay conditions. The protein concentration was determined by the method of Bradford (1976) with BSA as a standard. The molar concentration of Xyl II was calculated assuming an M_r value of 15,800 (Dey et al. 1992).

Denaturation/renaturation studies of xylanase

All denaturation and renaturation experiments were carried out in 50 mM potassium phosphate buffer, pH 7.2. Xyl II (100 μM) was denatured for 16 h in 6 M Gdn HCl. Renaturation was initiated by diluting 10 μL of the sample into a final volume of 1 mL of potassium phosphate buffer, pH 7.2. Next, 100-μL aliquots were withdrawn at various time intervals of refolding and assayed for Xyl II activity. Renaturation in the presence of α-crystallin was carried out at 37°C. After 1 h, ATP (0.3 mM) was added to the refolding solution and the activity was assayed and incubated further. The final concentrations of Xyl II and α-crystallin were 1 μM and 0.2 mg/mL, respectively. Renaturation of Xyl II in the presence of ATP analogs such as AMP, ATP, and AMP-PNP were also carried out.

Temperature-dependent refolding of Xyl II-α-crystallin complex

Renaturation of Xyl II was initiated at 37°C in the presence of α-crystallin. After 1 h, ATP (0.3 mM) was added to the complex and further incubated at different temperatures. Aliquots were removed at various time intervals and the enzyme activity was assayed.

Fluorescence studies

Fluorescence spectra were recorded with a Perkin-Elmer LS 50B spectrofluorometer equipped with a Julabo F25 water bath. The excitation and emission wavelengths are given in the figure legends. The fluorescence spectrum of α-crystallin was subtracted from the fluorescence spectrum of the complex. The resultant spectrum represents the fluorescence spectrum of the enzyme bound to α-crystallin. Additional experimental details are described in the text and figure legends.

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