©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Rapid Refolding Studies on the Chaperone-like -Crystallin
EFFECT OF alpha-CRYSTALLIN ON REFOLDING OF beta- AND -CRYSTALLINS (*)

(Received for publication, May 2, 1995; and in revised form, June 9, 1995)

Bakthisaran Raman Tangirala Ramakrishna Ch. Mohan Rao (§)

From the Centre for Cellular and Molecular Biology, Hyderabad 500 007, India

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

alpha-Crystallin, a multimeric protein present in the eye lens, is shown to have chaperone-like activity in preventing thermally induced aggregation of enzymes and other crystallins. We have studied the rapid refolding of alpha-crystallin, and compared it with other calf eye lens proteins, namely beta- and -crystallins. alpha-Crystallin forms a clear solution upon rapid refolding from 8 M urea. The refolded alpha-crystallin has native-like secondary, tertiary, and quaternary structures as revealed by circular dichroism and fluorescence characteristics as well as gel filtration and sedimentation velocity measurements. On rapid refolding, beta- and -crystallins aggregate and form turbid solutions. The presence of alpha-crystallin in the refolding buffer marginally increases the recovery of beta- and -crystallins in the soluble form. However, unfolding of these crystallins together with alpha-crystallin using 8 M urea and subsequent refolding significantly increases the recovery of these proteins in the soluble form. These results indicate that an intermediate of alpha-crystallin formed during refolding is more effective in preventing the aggregation of beta- and -crystallins. This supports our earlier hypothesis (Raman, B., and Rao, C. M.(1994) J. Biol. Chem. 269, 27264-27268) that the chaperone-like activity of alpha-crystallin is more pronounced in its structurally perturbed state.


INTRODUCTION

alpha-Crystallin is a multimeric protein, made up of the two homologous gene products of alpha(A) and alpha(B), present in high concentration in the eye lens. alpha(B) is also known to occur in non-lenticular tissues such as heart, muscle, and kidney(1, 2, 3, 4) . Its expression can be induced by thermal (5) or hypertonic stress(6) . It is structurally related to small heat shock proteins and behaves in several ways like the small Hsps(^1)(5, 7, 8, 9) . alpha-Crystallin is heat-stable up to 100 °C(10) .

Studies on alpha-crystallin over several years have been reviewed recently by Groenen et al.(11) . alpha-Crystallin has been considered as a structural protein; the quaternary structure of this crystallin has received much attention in order to gain an insight into its packing in the transparent eye lens. However, the understanding of the tertiary and quaternary structure of alpha-crystallin is still not complete. Identification of alpha-crystallin in several other tissues suggests that it might have a role to play in other functions. Like some Hsps(12, 13, 14) , it is known to interact with membrane (15, 16) and cytoskeletal elements (17, 18) and to modulate the intermediate filament assembly(19) . Mixed aggregates of alpha(B)-crystallin and small Hsps have been observed in vivo(20, 21) . alpha-Crystallin and Hsp25 form mixed aggregates in vitro and resemble each other in secondary structure and in stability toward urea denaturation(9) .

alpha-crystallin has been shown to prevent thermal aggregation of beta- and -crystallins and other proteins(22) . alpha(B)-Crystallin, like the small heat shock proteins Hsp25 and Hsp27, has been shown to facilitate the refolding of citrate synthase and alpha-glucosidase in vitro(23) . It forms a complex with carbonic anhydrase upon heat denaturation of the enzyme (24) . Recently, it has been shown to bind to ATP, but not to ADP or GTP (25) , and possess autokinase activity(26) . It is interesting to note that several years ago Bloemendal et al. showed that alpha- and beta-crystallins form a complex at high concentrations, whereas -crystallin remains uncomplexed(27) .

We had earlier investigated the chaperone-like activity of alpha-crystallin toward the photoaggregation of -crystallin (28) and the aggregation of insulin B chain upon reduction of interchain disulfide bonds of insulin(29) . It does not prevent aggregation of proteins at low temperatures but does so at temperatures above 30 °C. We have hypothesized that alpha-crystallin prevents aggregation of non-native structures by providing appropriately placed hydrophobic surfaces. A structural transition above 30 °C enhances the protective ability of alpha-crystallin, perhaps by increasing or reorganizing the hydrophobic surfaces. A structurally perturbed state, thus, is important in the chaperone-like activity of alpha-crystallin. We now report on the rapid refolding process of alpha-, beta-, and -crystallins in the context of the chaperone-like activity of alpha-crystallin. Our results suggest that an intermediate of alpha-crystallin, formed during its refolding, is substantially more effective in preventing the aggregation of beta- and -crystallins.


MATERIALS AND METHODS

Isolation and Purification of Crystallins

Fresh calf lenses were homogenized in Tris-HCl buffer, pH 7.2, containing 100 mM NaCl, 1 mM EDTA, and 0.02% sodium azide and centrifuged at 5000 g at 4 °C for 20 min. The soluble proteins in the supernatant were fractionated by gel filtration on a column of Bio-Gel A-1.5m (1.8 180 cm) at 4 °C. The fractions corresponding to beta(H)-, beta(L)-, and -crystallins were pooled and dialyzed against water and lyophilized. The lyophilized powders of these crystallins were stored at 4 °C. The fractions corresponding to alpha(L)-crystallin were pooled and concentrated at the above mentioned temperature by ultrafiltration using an Amicon ultrafiltration unit. The alpha(L) concentrate was further purified by gel filtration on Bio-Gel A-5m column (1.8 180 cm), and the fractions were pooled and concentrated by ultrafiltration as described above. The concentrate of purified alpha-crystallin was stored at -20 °C until required. We have noticed that lyophilization leads to alteration in its size; hence, we have not used lyophilized sample. It is important to note that the sample was kept at or below 4 °C during the entire isolation procedure in order to ensure that it remained in its high molecular weight form.

Unfolding and Rapid Refolding of Crystallins

Crystallins were dissolved in 20 mM phosphate buffer, pH 7.4, containing 8 M urea and equilibrated at room temperature for 5 h. Rapid refolding was achieved by diluting 50 µl of this sample with 950 µl of 10 mM phosphate buffer (pH 7.4) free of denaturant. The refolding buffer also contained 100 mM NaCl. The solution was vortexed for 30 s and equilibrated at room temperature for 1 h. Turbidity of the solutions was measured by monitoring the optical density at 500 nm in a Hitachi-330 spectrophotometer. The samples were centrifuged at 5000 g for 15 min, and the optical density at 280 nm of the supernatant was measured. The absorption coefficients (for 1 mg/ml) of 0.83, 2.3, 2.1, and 2.1 for alpha-(30) , beta(H), beta(L)-, and -crystallins(31) , respectively, were used for determination of their concentrations. In another experiment, 50 µl of the sample in denaturant (8 M urea) containing 0.3 mg of beta- or -crystallin was added to 950 µl of refolding buffer containing the required amount of alpha-crystallin. Rapid refolding of beta- or -crystallin together with alpha-crystallin was performed by adding 50 µl of the denatured protein sample containing 0.3 mg of beta- or -crystallin and varying amounts of alpha-crystallin, to 950 µl of the refolding buffer. The percentage recovery of the target protein was calculated from the A, assuming that recovery of alpha-crystallin is 100%. This assumption was verified in a separate experiment where only alpha-crystallin was refolded as mentioned earlier.

Fluorescence Spectroscopy

Fluorescence measurements were made on a Hitachi F4000 fluorescence spectrophotometer. Synchronous scanning fluorescence spectra were recorded as described by Rao(32) . The excitation and the emission monochromator were set at 250 and 290 nm, respectively, to get Delta of 40 nm, and both the monochromators were scanned simultaneously. Red edge excitation shift study was performed by taking fluorescence spectra of the native and refolded alpha-crystallin with excitation wavelengths of 280, 295, 300, and 305 nm. The excitation and emission bandpasses were set at 3 and 1.5 nm, respectively.

Circular Dichroism Measurements

Near UV and far UV CD spectra of the native and refolded alpha-crystallins were recorded on a Jasco J-20 spectropolarimeter. alpha-Crystallin refolded at 1 mg/ml was used for CD studies. Spectra were recorded using 0.05- and 2-cm path length cells for far UV and near UV regions, respectively.

Sedimentation Velocity Measurements

Sedimentation velocity measurements were done on 4 mg/ml native and refolded alpha-crystallin solutions using a Spinco E analytical ultracentrifuge fitted with a phase plate and rotor temperature indicator and control unit. Runs were performed at 68,000 rpm at an average temperature of 25 ± 0.5 °C.

s values were obtained by applying appropriate corrections for temperature and solvent viscosity.

Gel Filtration Experiments

Gel filtration experiments were performed using a Bio-Gel A-1.5m column (0.8 96 cm) equilibrated with Tris-HCl buffer, pH 7.2, containing 100 mM NaCl, 1 mM EDTA, and 0.02% sodium azide with a flow rate of 15 ml/h. Fractions (0.9 ml) were collected, and the optical density at 280 nm was monitored.


RESULTS AND DISCUSSION

Our understanding of protein folding, particularly in vivo, is far from complete despite the considerable progress made over the last two decades. According to the generally accepted paradigm, all the information required for protein folding resides in its primary sequence, and protein folding is an unassisted, spontaneous process. However, many proteins appear to require external assistance, mostly through other proteins, to fold and assemble to their functional forms. Chaperones that provide the required assistance (33, 34, 35) prevent the unfavorable interactions in the folding pathway of a protein. Hydrophobic interactions are known to play a crucial role in such molecular processes.

Recently we have shown that the chaperone-like activity of alpha-crystallin is enhanced when its quaternary structure is perturbed. In order to get a better insight into the chaperone-like activity of alpha-crystallin, we have studied the refolding properties of alpha-, beta-, and -crystallins using the rapid refolding method. This is one of the widely used methods (36, 37, 38) to study the refolding properties of a protein where the protein in its unfolded state is rapidly transferred to the condition(s) that favor its folding. In the present study, the crystallins were subjected to refolding at different concentrations. Usually proteins at high concentration tend to aggregate upon rapid refolding. Fig. 1A represents the turbidity profiles of crystallins as a function of the amount of protein taken for the refolding. Interestingly, alpha-crystallin remains clear upon rapid refolding in the concentration range studied, while beta- and -crystallins aggregate and yield turbid solution. Turbidity increases with concentration. We have observed that refolding of alpha-crystallin even at a concentration of 13 mg/ml yields a clear solution. The amount of protein recovered in the soluble form, after removing the precipitate by centrifugation, is shown in Fig. 1B. As evident from the figure, more than 90% of alpha-crystallin is recovered in the soluble form, while the other two crystallins kinetically partition to aggregate in a concentration-dependent manner. The recovery of -crystallin appears to be comparatively lower. This may be because -crystallin is more prone to aggregation or because of other factors such as preferential adsorption to the surface of the reaction tube.


Figure 1: Rapid refolding of alpha- (), beta(H)- (bullet), beta(L)- (), and - (up triangle, filled) crystallins. A, turbidity profile upon refolding at different concentrations of crystallins. Turbidity was measured as the optical density at 500 nm. B, variation of recovery of the crystallins in soluble form as a function of concentration. Percentage recovery of proteins is with respect to the amount taken for refolding. Concentrations of the crystallins are those obtained after dilution into refolding buffer.



Since the refolding of alpha-crystallin yields a clear solution, we performed CD and fluorescence spectroscopy to determine whether the refolded alpha-crystallin attains its native conformation. Fig. 2A shows the far UV CD spectra of native and refolded alpha-crystallin. These spectra overlap within the experimental error, suggesting that it regains almost all of its secondary structure. Comparison of near UV CD spectra of native and refolded alpha-crystallin (Fig. 2B) suggests that tertiary structure is also regained. It is also known that alpha-crystallin regains secondary and tertiary structures upon slow refolding (where the denaturant is removed by dialysis)(39, 40) .


Figure 2: Circular dichroism of native (-) and refolded (at 1 mg/ml)(- - -) alpha-crystallin. A, far UV CD spectra. B, near UV CD spectra.



The fluorescence emission maxima of native and refolded alpha-crystallins, upon excitation at 295 nm, do not differ; they are 336 and 336.6 nm, respectively. The synchronous spectrum of a mixture of fluorophores or fluorophore in different local microenvironment is likely to be more informative than the normal emission or excitation spectrum(41) . Synchronous scan might be expected to provide a signature and reflect minor structural perturbations. An earlier study on alpha-, beta-, and -crystallins using synchronous fluorescence scanning by one of us (32) suggests that the synchronous scan spectra of these crystallins are distinguishable and appear to provide a specific signature. The synchronous scan spectra of the native and refolded alpha-crystallin superimpose on each other as shown in Fig. 3, indicating that the microenvironments of the aromatic amino acids are indistinguishable in both cases.


Figure 3: Synchronous fluorescence spectra of native (-) and refolded(- - -) alpha-crystallin. Delta was set at 40 nm. Both excitation and emission band passes were set at 3 nm. I(f) = normalized fluorescence intensity (arbitrary units); = excitation wavelength (nm).



The shift in the fluorescence emission wavelength upon excitation with the wavelength of red edge of the absorption spectrum of a fluorophore is termed red edge excitation shift. This effect is usually observed for polar fluorophores in motionally restricted media(42, 43, 44, 45, 46, 47, 48) . Single-tryptophan proteins(46) , crystallins in intact eye lens and in solutions(47, 48) , have been studied using this technique. The magnitude of the shift in the observed emission maximum can be correlated to the restriction that the microenvironment imposes on the fluorophore. Both the native and the refolded alpha-crystallin show a 6-nm red edge excitation shift (336 to 342 nm) upon shifting the excitation wavelength from 295 to 305 nm. This indicates that the microenvironments around the tryptophans in the native and the refolded alpha-crystallin (or at least its influence on the mobility of the tryptophans) are identical.

Changes are known to occur in the quaternary structure of alpha-crystallin when it is subjected to temperature perturbation and on urea treatment(39, 49, 50, 51) . To find out the state of the quaternary structure of the refolded protein, we have performed gel filtration chromatography and sedimentation velocity measurement. Analysis of the elution profiles (Fig. 4) of the native and refolded alpha-crystallin suggests that the refolded alpha-crystallin might form a marginally smaller multimer compared with the native form. The sedimentation coefficient (s value) of the native alpha-crystallin, determined to be 17.1 S, is in good agreement with the reported value for the alpha(c) form(49) . The sedimentation pattern of the refolded alpha-crystallin shows a single peak (Fig. 5) with a S value of 16.1, suggesting that the size of the refolded alpha-crystallin is marginally smaller than that of the native alpha-crystallin. Packing alterations, if any, without significant shape and size changes might not be detected in the sedimentation studies.


Figure 4: Elution profile of native () and refolded (bullet) alpha-crystallin on a Bio-Gel A-1.5m column (see ``Materials and Methods'' for details). The amount of protein loaded was 1 mg in both cases. The elution positions of blue dextran (2000 kDa) (A); thyroglobulin (670 kDa) (B); and lysozyme (14.4 kDa) (C) are indicated by arrows.




Figure 5: Sedimentation pattern of native (A) and refolded (B) alpha-crystallin (see ``Materials and Methods'' for details).



These results indicate that alpha-crystallin regains most of its three-dimensional structure upon rapid refolding and that it does not need any external assistance. It is noteworthy that RNase A, which is known to refold completely to its active form at low concentrations (<0.1 mg/ml), misfolds and aggregates upon refolding at concentrations higher than 0.1 mg/ml(38) . alpha-Crystallin, on the other hand, refolds to its native state even at concentrations as high as 13 mg/ml. This is significant and might be relevant to its chaperone-like activity.

Several proteins need the assistance of molecular chaperones to fold correctly to their native state. Whether chaperones themselves need such assistance is not clear. Few studies have attempted to answer this question. Chemically synthesized GroES not only folds and assembles properly but also shows complete activity in refolding of rubisco in the presence of GroEL(52) . Urea-unfolded GroES regains its native assembly upon removal of the denaturant(52) . GroEL, dissociated to its monomeric form by urea, needs Mg-ATP to reassemble to its native polymeric form, and the presence of native GroEL assembly enhances the process of such reassembly(53) . Newly synthesized mitochondrial chaperonin Hsp60 monomers do not assemble in the absence of preexisting native protein assembly(54) . These studies have led to the concept of ``self-chaperoning.'' Thus, it appears that heat shock proteins and chaperones have the tendency to fold spontaneously to their native structure without any external assistance, and some multimeric chaperones such as GroEL chaperone their own assembly. The mechanism by which alpha-crystallin refolds properly is not known. The possibilities are: (i) the individual subunits refold and then assemble to form large structures; (ii) the domains of each subunit that need to interact for multimerization refold first, and subsequently the other domains fold; and (iii) both folding and multimerization occur simultaneously. Further studies are required to verify whether there is a general preference for any of these possibilities.

As stated earlier, -crystallin undergoes photoaggregation, and alpha-crystallin prevents this aggregation by forming a complex(28) ; it is also known to prevent the thermal aggregation of beta- and -crystallins(22) . Our results (Fig. 1) show that rapid refolding of beta- and -crystallins also leads to aggregation and turbidity. We have, therefore, studied the effect of alpha-crystallin on this aggregation of beta- and -crystallins. The recovery of these proteins in the soluble form increases to some extent when alpha-crystallin is present in the refolding buffer. The recovery of these proteins with the increasing concentration of alpha-crystallin is shown in Fig. 6. The increase in the alpha-crystallin concentration marginally enhances the recovery of beta- and -crystallin as shown in the figure. Even a 10-fold excess of alpha-crystallin does not improve the recovery of -crystallin to more than 25-30%.


Figure 6: Refolding of beta(H)- (bullet), beta(L)- (), and - () crystallins in the presence of native alpha-crystallin. The percentage recovery of protein is with respect to the amount of protein taken for refolding, which is 0.3 mg. The amount of alpha-crystallin in the refolding buffer was varied to get different [alpha]/[p] (w/w) ratios. [p] = Concentration of beta- or -crystallin.



Interestingly, when beta- or -crystallins are denatured together with alpha-crystallin and then subjected to refolding, the recovery of beta- or -crystallin in the solution is increased significantly. Fig. 7A represents the turbidity profile of solutions obtained by refolding of 0.3 mg of beta- or -crystallin together with increasing amounts of alpha-crystallin; the percentage recovery of these proteins is shown in Fig. 7B. Even at a 5 times lesser concentration (w/w), alpha-crystallin is able to almost completely prevent the aggregation of beta-crystallin. Recovery of -crystallin changes from 20 to 60% ( Fig. 6and Fig. 7B). These results suggest that the native alpha-crystallin is less effective in preventing the aggregation of beta- or -crystallin. On the other hand, when alpha-crystallin is refolded together with beta- or -crystallin, it prevents the aggregation of beta- or -crystallin very significantly. We believe that an intermediate state (or assembly) of alpha-crystallin formed in its refolding pathway is more effective in preventing the aggregation of the other two crystallins when compared with the native protein. In other words alpha-crystallin is more functional in its structurally perturbed state in preventing the aggregation of other crystallins. This supports our hypothesis that alpha-crystallin prevents aggregation of non-native structures by providing appropriately placed hydrophobic surfaces and that a structural perturbation enhances the protective ability of alpha-crystallin. The structurally perturbed state, thus, is important in the chaperone-like activity of alpha-crystallin.


Figure 7: Refolding of beta(H)- (bullet), beta(L)- (), and - () crystallins together with alpha-crystallin. A, turbidity profile upon refolding of mixtures of alpha- and beta-crystallins or alpha- and -crystallins at different ratios. Turbidity was measured as optical density at 500 nm. B, recovery of beta- or -crystallins in soluble form as a function of [alpha]/[p] ratios. The percentage recovery of protein is with respect to the amount of protein taken for refolding, which is 0.3 mg. The amount of alpha-crystallin was varied to get different [alpha]/[p] (w/w) ratios. [p] = Concentration of beta- or -crystallin.



On the basis of our results, we conclude that alpha-crystallin rapidly refolds and regains most of the secondary, tertiary, and quaternary structures. This might be a common property of chaperones and heat shock proteins. The other eye lens crystallins such as beta- and -crystallins aggregate and precipitate out upon such rapid refolding. Native alpha-crystallin prevents this aggregation to a lesser extent. However, if beta- or -crystallin is denatured together with alpha-crystallin and then subjected to the refolding process, alpha-crystallin almost completely prevents the aggregation of other crystallins, suggesting the importance of a structurally perturbed state. These results should prove useful in understanding intercrystallin interactions as well as the mechanism of the chaperone-like activity of alpha-crystallin.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Centre for Cellular and Molecular Biology, Hyderabad 500 007, India. Tel.: 91-40-672-241; Fax: 91-40-671-195; mohan{at}ccmb.uunet.in.

(^1)
The abbreviation used is: Hsp, heat shock protein.


ACKNOWLEDGEMENTS

We thank V. V. S. N. Sarada for help in the initial experiments. We thank Shri. Pramod for technical help in sedimentation velocity measurements.


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