Subunit Exchange Demonstrates a Differential Chaperone Activity of Calf alpha -Crystallin toward beta LOW- and Individual gamma -Crystallins*

Tatiana Putilina, Fériel Skouri-Panet, Karine Prat, Nicolette H. LubsenDagger , and Annette Tardieu§

From the Laboratoire de Minéralogie-Cristallographie, CNRS and P6-P7 Universities, Case 115, 4 Place Jussieu, F75252 Paris Cedex 05, France and the Dagger  Department of Biochemistry 161, University of Nijmegen, P. O. Box 9101, 6500 HB Nijmegen, The Netherlands

Received for publication, August 9, 2002, and in revised form, January 30, 2003

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The chaperone activity of native alpha -crystallins toward beta LOW- and various gamma -crystallins at the onset of their denaturation, 60 and 66 °C, respectively, was studied at high and low crystallin concentrations using small angle x-ray scattering (SAXS) and fluorescence energy transfer (FRET). The crystallins were from calf lenses except for one recombinant human gamma S. SAXS data demonstrated an irreversible doubling in molecular weight and a corresponding increase in size of alpha -crystallins at temperatures above 60 °C. Further increase is observed at 66 °C. More subtle conformational changes accompanied the increase in size as shown by changes in environments around tryptophan and cysteine residues. These alpha -crystallin temperature-induced modifications were found necessary to allow for the association with beta LOW- and gamma -crystallins to occur. FRET experiments using IAEDANS (iodoacetylaminoethylaminonaphthalene sulfonic acid)- and IAF (iodoacetamidofluorescein)-labeled subunits showed that the heat-modified alpha -crystallins retained their ability to exchange subunits and that, at 37 °C, the rate of exchange was increased depending upon the temperature of incubation, 60 or 66 °C. Association with beta LOW- (60 °C) or various gamma -crystallins (66 °C) resulted at 37 °C in decreased subunit exchange in proportion to bound ligands. Therefore, beta LOW- and gamma -crystallins were compared for their capacity to associate with alpha -crystallins and inhibit subunit exchange. Quite unexpectedly for a highly conserved protein family, differences were observed between the individual gamma -crystallin family members. The strongest effect was observed for gamma S, followed by hgamma Srec, gamma E, gamma A-F, gamma D, gamma B. Moreover, fluorescence properties of alpha -crystallins in the presence of bound beta LOW-and gamma -crystallins indicated that the formation of beta LOW/alpha - or gamma /alpha -crystallin complexes involved various binding sites. The changes in subunit exchange associated with the chaperone properties of alpha -crystallins toward the other lens crystallins demonstrate the dynamic character of the heat-activated alpha -crystallin structure.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

alpha -Crystallins and small heat shock proteins have in common a conserved C-terminal domain of about 115 residues, the alpha -crystallin domain, the structure of which is organized around an eight-stranded beta -sandwich (1, 2). The small heat shock proteins usually associate into high molecular weight monodisperse or polydisperse oligomers, able to protect against stress through the binding of a variety of partially unfolded substrates. alpha -Crystallin was demonstrated by Horwitz in 1992 (3) to also exhibit chaperone properties in vitro. It is able to bind beta - and gamma -crystallins at the onset of their thermal denaturation, thus preventing further denaturation and aggregation, yet not refolding. Following this pioneering work, the chaperone-like activity of alpha -crystallin has been the subject of numerous studies and functional models have been suggested (4-30). Essentially, hydrophobic patches on the surface, either present in the native state or revealed after structural modifications, would associate with a variety of partially denatured substrates from insulin or alpha -lactalbumin to the other lens proteins, in a substrate-dependent manner. Moreover, the dynamic character of the alpha -crystallin quaternary structure with the occurrence of temperature dependent subunit exchange was demonstrated and analyzed (31, 32). Fluorescence resonance energy transfer (FRET)1 was found particularly useful to demonstrate that subunits, from recombinant alpha A- or alpha B-crystallin, can also reversibly exchange between oligomers (31, 33-36). Subunit exchange was also detected for other small heat shock proteins such as Hsp27, Hsp16.9, and Hsp16.5 (2, 37). The studies also demonstrated that subunit exchange might be partially inhibited in the presence of the bound substrates.

In the physiological context of the lens, such properties of alpha -crystallins would be advantageous in protecting against cataract by delaying the formation of light-scattering aggregates, which affect lens transparency. Yet, only a few chaperone studies with beta - and gamma -crystallins as substrates have been performed (4, 5, 38-41) without any correlation with subunit exchange and without any attempt to analyze possible differences between different members of the B-gamma -crystallin family. It is therefore the aim of this paper to further investigate these points.

This study was performed with native calf crystallins and one human recombinant gamma S (hgamma Srec). Some of our preliminary experiments and recently published data (42-44) indicated that, at the high temperatures necessary for the chaperone activity of alpha -crystallin toward other lens proteins, an important conformational change in alpha -crystallin structure takes place. Therefore, the temperature-induced structural changes in alpha -crystallin were analyzed in relation to its subunit exchange properties. Possible differences in alpha -crystallin behavior toward different lens proteins as substrates were evaluated by FRET using IAEDANS- and IAF-labeled subunits. The rates of alpha -crystallin subunit exchange were compared depending on bound beta gamma -crystallins.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- 5-(Iodoacetamido)fluorescein (5-IAF), 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid (1,5-IAEDANS), tris-(2-cyanoethyl)phosphine, and tris-(2-carboxyethyl)phosphine, hydrochloride were purchased from Molecular Probes (Eugene, OR). All other chemicals were of analytical grade from Sigma.

Purification of Calf Lens Crystallins-- The alpha -, beta -, and gamma -crystallins were prepared from young calf lenses as described before (45, 46). Briefly, the cortex was separated from the nucleus by a gentle stirring of the lens in 150 mM phosphate buffer, pH 6.8 (22 mM Na2HPO4, 28 mM KH2PO4, 200 mM KCl, 1.3 mM EDTA, 3 mM NaN3, 3 mM dithiothreitol) for 15-30 min at 6 °C. The alpha -, beta LOW-, and cortical gamma - (gamma C) crystallins were prepared from the clear supernatant fraction of the cortical extracts by gel filtration, using a Amersham Biosciences FPLC system and a Superdex S-200 PG column. The nuclear gamma -crystallins (gamma N) were purified in the same way from the nuclear extracts. The individual gamma -crystallins were further purified by cation exchange chromatography on a Mono S HR 10/10 column. Crystallin concentrations were calculated using molar extinction coefficient of 0.85 cm2 mg-1 for alpha -crystallin, 2.3 cm2 mg-1 for beta LOW-, 1.85 cm2 mg-1 for gamma S-, 2.05 cm2 mg-1 for gamma E-, 2.1 cm2 mg-1 for gamma D-, 2.02 cm2 mg-1 for gamma B-, and 2.0 cm2 mg-1 for gamma A-F-crystallin (that elute together).

Expression and Purification of Human gamma S-crystallin-- The recombinant human gamma S (hgamma Srec) was prepared as described previously (47). In short, the coding sequence was amplified from a human lens cDNA library and cloned NdeI/BamHI in the pET3a vector. The expression construct was introduced into the BL21(DE3) pLysS bacterial strain, and protein expression was induced by addition of isopropyl-beta -D-thiogalactopyranoside. The protein was extracted from cells using standard procedure and purified by gel filtration using a Sephacryl S200 HR column. The purity of the recombinant protein was checked by SDS-PAGE electrophoresis and mass spectrometry.

Chaperone Activity Assays-- The chaperone-like activity of alpha -crystallin was monitored by measuring the absorbance at 360 nm as a function of time. Measurements were made every minute during half an hour with a UV/VIS LambdaBio spectrophotometer. The concentration of alpha -crystallin remained at 20 mg/ml and beta gamma -crystallins were added at different w/w ratios. The temperature inside the cuvette was thermostated with a Ministat water bath and was verified prior to data collection using a Checktemp 1 microthermocouple thermometer. The soluble fractions were examined by gel filtration through a Sephacryl S200 HR column, using a Amersham Biosciences FPLC system.

Gel Electrophoresis and Western Blot Analysis-- Samples were prepared by mixing proteins v/v with the buffer A (125 mM Tris, pH 6.8, 20% glycerol, 4% SDS, 1% beta -mercaptoethanol) and boiling (48). SDS-PAGE fractionation was performed on 15% polyacrylamide gels. Proteins were blotted to polyvinylidene difluoride membrane (Bio-Rad) by capillarity in the buffer B (10 mM Tris pH8.2, 2 mM EDTA, 50 mM NaCl, 2 mg/l dithiothreitol) and immunoreactions were carried out as described in the immunoblotting kit manual (Anti-rabbit IgG, Alkaline Phosphatase; Calbiochem) with anti-gamma -crystallin polyclonal antibodies at dilution 1:500.

Small Angle X-ray Scattering (SAXS)-- The intensity scattered by one particle as a function of the scattering vector s (where s = 2sintheta /lambda and 2theta is the scattering angle), usually called the particle form factor, is the Fourier transform of the spherically averaged autocorrelation function of the electron density contrast associated with the particle. When the solution is ideal, i.e. in the absence of interactions, the total scattered intensity, I(s), is the sum of the scattering of the individual particles, and the scattering near the origin can be written (49): I(s) = exp[-4/3pi 2R<UP><SUB><IT>g</IT></SUB><SUP>2</SUP></UP>s2]; therefore, "Guinier plots," i.e. plots of log I(s) versus s2, provide us with the extrapolated intensity at the origin, I(0), which is proportional to the molecular weight and with the radius of gyration, Rg, which is a function of the particle size. The experiments were carried out using the small angle instrument D24 using the synchrotron radiation emitted by the storage ring DCI at the Laboratory for Synchrotron Radiation, L.U.R.E. (Orsay, France). Data were collected using a linear position-sensitive detector with a delay line readout. The s-increment per channel was 0.000146 Å-1, and the average recorded s range was 0.2 × 10-2 < s < 4 × 10-2 Å-1. The wavelength of the x-rays was 1.488 Å (K-edge of nickel). The solution experiments were performed with a specially designed quartz cell operated under vacuum (50) that could be filled and rinsed in situ. The intensity curves, I(c,s), were subtracted for background and, within each series, scaled on the same relative value with a normalization for concentration. No scaling was done between series.

Labeling of Crystallins with Fluorescent Probes-- The labeling reaction was carried out at a final protein concentration of about 1 mg/ml in 150 mM phosphate buffer, 10 mM EDTA, pH 6.8, following the same protocol for both probes. Prior to labeling, a 10-fold molar excess of each reducing agents tris-(2-carboxyethyl) phosphine hydrochloride and tris-(2-carboxyethyl)phosphine were added to assure the reduction of disulfide bonds. Each probe was solubilized in methanol, and a small aliquot was added for a final ratio of 20 mol of probe/mol of protein. The reaction then proceeded for 4 h at 37 °C, followed by overnight incubation at 4 °C. The labeling was terminated by the addition of a 10-fold molar excess of mercaptoethanol. A vacuum concentrator unit with 100,000 and 10,000 cut-off membranes were used to remove any unconjugated probe until no fluorescence could be detected in the eluate for alpha - and gamma -crystallin, respectively. The molar ratio of attached probe was calculated from the absorbance spectrum of the labeled protein and that of a free probe. Typically, all Cys residues in alpha -crystallin could be labeled with either probe. For gamma -crystallin two residues per molecule could be modified with IAEDANS.

Steady State Fluorescence-- Fluorescence measurements were made with SPF-25 spectrofluorometer using quartz cuvettes with a 10-mm path length. Excitation and emission slits were set to 5-nm bandwidth. Data were corrected for the contribution of solvents. For temperature dependence studies, the cells were thermostated with a Ministat water bath. The temperature was verified within each cuvette prior to data collection using a Checktemp 1 microthermocouple thermometer. All samples were in phosphate buffer, pH 6.8, unless specified. Microcuvettes with stopper were used for incubation at high temperatures to avoid evaporation.

Subunit Exchange-- Experiments on subunit exchange of alpha -crystallin were based on the protocol of Bova et al. (31) with the following modifications. Two populations of alpha -crystallin labeled with 1,5-IAEDANS (donor) or 5-IAF (acceptor) were heated at 60 or 66 °C for 30 min, either alone or in the presence of other crystallins, and all samples were returned to room temperature afterward. The rate of subunit exchange was evaluated from energy transfer data. Donor and acceptor labeled alpha -crystallins were mixed at 37 °C, and the degree of energy transfer was estimated over time by following the decrease in the intensity of the donor emission. Wavelengths of excitation and emission were 335 and 460 nm. The rate was calculated from the equation F(t)/F(0) = A + Be-kt, where k is defined as the transfer rate constant, and F(t)/F(0) corresponds to the emission intensity ratio at time = t and 0, respectively (34). The efficiency of energy transfer (E) at different time points was calculated from the relationship to the intensity of the donor fluorescence in the absence (Fa) and presence (Fd) of the acceptor: E = 1 - (Fd/Fa).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Labeling of alpha -Crystallin with Fluorescent Probes-- Two populations of alpha -crystallins were labeled covalently at the cysteine residues with either IAEDANS or IAF. IAEDANS and IAF are fluorescent probes that have been well characterized over the past years (51, 52). They are of relatively small size and are not known to induce conformational changes upon binding to proteins. Molar extinction coefficients of 5700 (IAEDANS), 75,000 (IAF), and 0.8 (alpha -crystallin, 1 mg/ml) were used to estimate the molar ratio of the covalently attached probe. Calf alpha -crystallin oligomers consist of alpha A (2/3) and alpha B subunits (1/3). alpha B has no cysteine residues but all cysteine residues at a single site at Cys-131 (beginning of strand beta 8 according to (1)) on alpha A subunit could be labeled. The integrity of labeled alpha -crystallin in our experiments was confirmed by unchanged tryptophan fluorescence and accessibility to acrylamide (results not shown). Labeling of gamma S (molar extinction coefficient, 1.85) with IAEDANS resulted in the addition of two fluorophore molecules per one gamma S, i.e. some of the five Cys residues could be modified. IAEDANS are preferentially reactive with cysteine residues in hydrophobic environment, and the labeling is in agreement with common structural features of gamma -crystallin.

alpha -Crystallin Quaternary Structure at High Temperature as Studied by SAXS-- The method that we have been using for a long time to purify alpha -crystallin from cortical extracts of calf lenses reproducibly provided us with oligomers that we characterized by SAXS to be made up of about 40-45 subunits, with a radius of gyration of about 62 Å and an external diameter of 170Å (45, 53). These values are in agreement with the molecular weight values usually reported in the literature and with recent electron microscopy (EM) observations (43). Yet, alpha -crystallin quaternary structure is known for a long time to be sensitive to environmental conditions (53). Moreover, our preliminary investigations and recently published studies (42, 43) have shown that, at the high temperatures required for the chaperone effect of alpha -crystallin toward other lens proteins to take place (60 °C for the beta - and 66 °C for the gamma -crystallin), the alpha -crystallin quaternary structure undergoes a major, irreversible, conformational change. We therefore used SAXS to further investigate the point. Purified fractions of alpha -crystallin were incubated at 60 or 66 °C for various periods of time, and at different protein concentrations, from 5 to 20 mg/ml. The samples were then cooled either to room temperature or to 4 °C, controlled by gel filtration, and analyzed by SAXS. Typical results are shown in Fig. 1. The gel filtration profile in Fig. 1a obtained after 1 h of incubation at 60 °C indicates that all the native alpha -crystallins have been transformed into higher molecular weight particles, since they elute in the void volume. SAXS experiments performed at 60 °C (Fig. 1b) provided us with quantitative values. The conformational change is achieved within about 10 min, and no reversibility whatsoever is observed upon return to room temperature (not shown). The 60 °C molecular weight is about twice the native one. The shape of the scattering curve also indicates that the alpha -crystallin size has increased. The increase in both size and molecular weight is even more pronounced after incubation at 66 °C (Fig. 1b). The radii of gyration of native and of 60 and 66 °C incubated samples were, respectively, 61.4, 82.3, and 98 Å. These observations are in agreement with the EM data that show much bigger particles after incubation at high temperatures (43). The shape of the SAXS intensity curves at low angles, however, is not consistent with the presence in our experimental conditions of chain-like structures, as seen by EM (43). Experiments performed with different incubation times (not shown) indicate that the high temperature quaternary structure remains stable for at least 1 h. Yet, the molecular weight and size may continue to grow, albeit slowly, with prolonged incubation time, e.g. overnight incubation at 66 °C usually leads to whitish precipitates.


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 1.   The chaperone-like activity of alpha -crystallin at elevated protein concentrations. a, gel filtration profile of alpha -crystallin at room temperature before (diamond ) and after (black-diamond ) incubation for 1 h at 60 °C; b, x-ray scattering intensities of alpha -crystallin (10 mg/ml) at room temperature (open circle ), 60 °C (black-diamond ), 66 °C (t); c, gel filtration profile of alpha  and beta LOW mixtures (2:1, w:w) at room temperature before (diamond ) and after (black-diamond ) incubation at 60 °C for 60 min; d, x-ray scattering intensities for alpha -crystallin (10 mg/ml) in the absence (, room temperature; black-square, 60 °C) and presence (open circle , room temperature; , 60 °C) of beta LOW (2:1, w:w); e, gel filtration profile of alpha - and gamma C-crystallin mixtures (4:1, w:w) at room temperature before (diamond ) and after (black-diamond ) incubation at 66 °C for 60 min; f, x-ray scattering intensities of alpha -crystallin (diamond , room temperature; black-diamond , 66 °C), alpha - and gamma C, 4:1, w:w (down-triangle, room temperature; black-down-triangle , 66 °C), alpha - and gamma N, 4:1, w:w (triangle , room temperature; black-triangle, 66 °C); g, temperature effect on the aggregation state of different crystallins followed by the absorbance (360 nm) at 60 °C for alpha -crystallin (diamond ), beta LOW (down-triangle), gamma N (open circle ), gamma C (), and at 66 °C for alpha - (black-diamond ) and gamma N (); h, chaperone activity assay for different mixtures of lens crystallins. The absorbance at 360 nm was recorded during the incubation of alpha  with beta LOW (2:1, w:w, 60 °C, ), with gamma N(4:1, w:w, 66 °C, black-down-triangle ), with gamma C (4:1, w:w, 66 °C, down-triangle); i, SDS gel electrophoresis: lane 1, alpha -crystallin; lane 2, gamma C-crystallin; lane 3, alpha - and gamma C-crystallin mixture at room temperature; lane 4, alpha  and gamma C high molecular weight peak (Fig. 1e). k, Western blot of the same samples, using gamma -crystallin antibodies; positive staining in high molecular fraction (lane 4) confirms the association of gamma - and alpha -crystallins.

Temperature Effect on Lens Crystallins as Observed by Fluorescence-- The effect of temperature on lens crystallins was also evaluated by steady state fluorescence techniques. The decrease in the quantum yield of tryptophan fluorescence with increase in temperature was observed for all crystallins studied (Fig. 2). Yet, the overall decrease did not reach the maximum possible for free tryptophan in solution (Fig. 2g). Together with the fact that the maxima of Trp fluorescence were only slightly red shifted, this indicates that all crystallins essentially retain their tertiary structure at temperatures up to 66 °C. The result is in agreement with infrared spectroscopy studies, which show that secondary structures are essentially preserved (39). On return to ambient temperatures, however, the changes were not totally reversible reflecting different stability of the lens proteins (Fig. 2). alpha -Crystallin displayed an increase in quantum yield, with the maximum of emission returning to 333 nm (Fig. 2a). beta LOW-Crystallin displayed a larger increase in quantum yield, and the red shift in maximum of emission was not reversible (Fig. 2b), whereas gamma -crystallin exhibited good reversibility in Trp emission (Fig. 2, c and d). All experiments using fluorescence techniques were conducted at concentrations below 0.05 mg/ml, and in contrast to the high protein concentration observations, no insoluble precipitated beta LOW- or gamma -crystallin could be detected after incubation at 60 or 66 °C for up to 30 min. However the appearance of a Rayleigh peak at 55 °C for beta LOW-crystallin and at 66 °C for gamma -crystallin was an indication of light scattering by larger, soluble, protein aggregates (Fig. 2). Such a difference in the onset of the Rayleigh peak reflects differential responses to heat stress among gamma - and beta LOW-crystallins.


View larger version (35K):
[in this window]
[in a new window]
 
Fig. 2.   Spectral properties of lens proteins at different temperatures. Fluorescence spectra were recorded using tightly closed cuvettes after incubation for 30 min at 37 °C (black-diamond ), 60 °C (), 66 °C (o), and 37 °C on return from high temperature (diamond ). Concentrations of proteins were adjusted to ensure the absorbance below 0.05 at the wavelength of excitation. Monochromator bandwidth of 5 nm was used in all experiments. a, alpha -crystallin; b, beta LOW-crystallin; c, gamma S-crystallin; d, gamma B-crystallin; e, alpha -IAF; f, alpha -IAEDANS; g, L-tryptophan as control in phosphate buffer; h, normalized emission spectrum of alpha -IAEDANS, lambda ex = 335 nm (triangle ), and the excitation spectrum of alpha -IAF, lambda em = 550 nm (down-triangle). The overlap between spectra illustrates the feasability of FRET between labeled molecules. Emission spectra of protein tryptophan fluorescence were collected using lambda ex = 295 nm; emission spectra of alpha -IAEDANS and alpha -IAF were collected using lambda ex = 335 nm and lambda ex = 460 nm.

A Rayleigh peak is also observed with alpha -crystallin at the onset of the conformational change at 60 °C. Moreover, changes in extrinsic fluorescence of labeled alpha -crystallin cysteine residues are illustrated by emission spectra of alpha -IAEDANS and alpha -IAF at 37 °C, before and after incubation at 66 °C for 30 min (Fig. 2, e and f). As can be seen from Fig. 2f, the incubation of alpha -crystallin leads to a decrease in hydrophobicity around cysteine residues that is only partially reversible after the return to ambient temperature. Such an increase is consistent with an increase in molecular size. Because of its small Stokes shift (spectral distance between absorption and emission maxima), the fluorescence of fluorescein compounds is known to be quenched in proteins by homotransfer (51). The overall increase in the average distance distribution between labeled cysteines would release the quench, resulting in increased fluorescence yield. Overall, the data on alpha -crystallin are consistent with minor changes in the secondary and tertiary structure of the subunits and with the size increase that has been demonstrated by SAXS.

The Chaperone Activity of alpha -Crystallins Is Associated with the Formation of Heterocomplexes-- The chaperone-like activity of the alpha -crystallin versus beta LOW- and gamma -crystallin, i.e. the ability to prevent temperature induced precipitation of the beta LOW/gamma -crystallin, was studied at 60 and 66 °C, respectively, by measuring the absorbance as a function of time at 360 nm (3, 4), with protein concentrations varying from about 2 mg/ml up to 20 mg/ml. Most of the assays reported in the literature have been performed at protein concentrations of about or below 0.5 mg/ml. The use of much higher protein concentrations would be more relevant to the in vivo conditions. It allowed us to confirm the chaperone activity of alpha -crystallin at elevated protein concentrations as well (Fig. 1h). Gel filtration analysis and SAXS studies were also performed at the same high protein concentrations facilitating the comparison of all data on chaperone activity. In this study the total gamma -crystallin extracts gamma N (from nucleus) or gamma C (from cortex) were used. The beta LOW-, gamma N-, and gamma C-crystallin alone were found to precipitate rapidly, in 5-15 min (Fig. 1g). We indeed found, as already reported in the literature, that alpha -crystallin easily prevents beta LOW-crystallin aggregation for at least one hour. The same concentrations of alpha -crystallin, however, had a somewhat different chaperone effect on gamma N/C-crystallin. As seen in Fig. 1h, alpha -crystallin is a less effective chaperone for gamma N/C-crystallin, and although the light scatter could be offset in the presence of alpha -crystallin, precipitation nevertheless occurred as a function of time, even when using increasing amounts of alpha -crystallin (4:1, w:w ratio, Fig. 1h).

The irreversible association of beta LOW- to alpha -crystallin at high protein concentrations (>= 10 mg/ml) was demonstrated for the first time by a combination of gel filtration, SDS gel electrophoresis (not shown), and SAXS as seen in Fig. 1, c and d. After 30-min incubation at 60 °C, the gel filtration alpha -crystallin peak is displaced toward higher molecular weight values, whereas the beta LOW-crystallin peak disappears (Fig. 1c) in agreement with the formation of soluble alpha /beta LOW aggregates. The association was easily shown by the SAXS experiments. At room temperature the intensity curves for the alpha /beta LOW mixture corresponded to the sum of the intensity curves recorded for alpha - and beta -crystallin alone. At 60 °C, the scattering of the beta -crystallin alone cannot be recorded since the sample precipitates. Yet, after incubation at 60 °C, the scattering at low angles for the alpha /beta LOW mixture was higher than the scattering for the high temperature form of alpha -crystallin indicating an increased average molecular weight and therefore the association of beta LOW- and alpha -crystallin, Fig. 1d).

The demonstration of an association between alpha - and gamma N/C-crystallin was less straightforward, since at high protein concentration (10 mg/ml) the mixture precipitates as a function of time. Analysis of the supernatant by gel filtration showed that part of the gamma -crystallin fraction still eluted at the same place (Fig. 1e), while by SAXS no significant aggregation could be observed (Fig. 1f). To clarify this point, SDS gel electrophoresis and Western blot analysis of the "alpha -crystallin peak" were performed (Fig. 1, i and k) using gamma C-crystallin and specific antibody. The experiments demonstrated that gamma C/alpha -crystallin complexes were present, but that at high protein concentration, once associated, the alpha - and gamma C-crystallin co-precipitate rapidly.

The association between alpha - and gamma -crystallin was confirmed for protein concentrations below 0.05 mg/ml by FRET. In these experiments mixtures of IAEDANS-labeled gamma S-crystallin (donor) and IAF-labeled alpha -crystallin (acceptor) were incubated in closed tubes at 66 °C for 10, 20, and 30 min. Emission spectra of each sample were recorded and compared with spectra at 25 °C before and after incubation. As seen from Fig. 4a there is a progressive decrease in donor fluorescence (460 nm), indicating the gamma S/alpha -crystallin association at 66 °C with an estimated efficiency of transfer of about 0.15 after 30 min. Most importantly, the binding was irreversible, as the decrease in donor fluorescence remained after the return to ambient temperature. For comparison the emission of donor in the absence of acceptor was unaffected by incubation at 66 °C (not shown). No energy transfer could be detected for gamma S- and alpha -crystallin mixtures incubated at 25 °C or 37 °C.

Native alpha -Crystallin Subunit Exchange-- The subunit exchange in recombinant alpha A-crystallin had been demonstrated previously by FRET (31, 33, 34). Here, we present data on the subunit exchange in native calf alpha -crystallin, containing both alpha A (75%) and alpha B (25%) subunits. Subunit exchange was studied at room temperature and 37 °C when alpha -crystallin consists of 40-45 subunits on average, at 60 °C and at 66 °C when the average number of subunits is 80-100 (Fig. 3, a and b). As can be seen on Fig. 3b the time course of subunit exchange in alpha -crystallin is increasing with the increase in temperature and is too fast to be reliably measured above 60 °C. Similar dependence on temperature had been reported for alpha A-crystallin using other fluorescent probes (34). The transfer rate constant at 37 °C was estimated to be 6.33 × 10-4 s-1, in agreement with the average value of 6.36 × 10-4 s-1 published for recombinant alpha A-crystallin (34). Also, the energy transfer could be reversed by the addition of unlabeled alpha -crystallin (34). Therefore, it seems that the mechanism of subunit exchange in native or recombinant crystallins is the same. We then compared the subunit exchange at 37 °C before and after half an hour incubation at 60 or 66 °C, i.e. after the heat-induced alpha -crystallin structural change. As can be seen from Fig. 3c the preincubation of alpha -crystallin at either 60 or 66 °C does not prevent subsequent subunit exchange at 37 °C. On the contrary, the rate for subunit exchange at 37 °C for heat-treated alpha -crystallin is significantly higher than for untreated proteins. It therefore appears that the temperature-induced structural changes lead to a more flexible and dynamic alpha -crystallin structure, able of more rapid subunit exchange.


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 3.   Subunit exchange in alpha -crystallin. alpha -IAEDANS (0.4 mg/ml) and alpha -IAF (0.4 mg/ml) were incubated at 60 °C for 30 min prior to return to the ambient temperature. These samples were used for subunit exchange at 37 °C: a, equal volumes of alpha -IAEDANS and alpha -IAF were mixed, diluted to 0.05 mg/ml, and a series of spectra were recorded (lambda ex = 335 nm) over a period of 2 h (diamond , 0 min; , 10 min; down-triangle, 20 min; open circle , 30 min; triangle , 60 min; black-diamond , 120 min). b, alpha -IAEDANS (0.4 mg/ml) and alpha -IAF (0.4 mg/ml) were preincubated each with an equal volume of beta LOW (0.4 mg/ml) at 60 °C for 30 min prior to return to ambient temperature. Subunit exchange was monitored at 37 °C as described above for the alpha -crystallin alone (a). c, effect of temperature on the subunit exchange. Subunit exchange between alpha -IAEDANS and alpha -IAF at 15 °C (diamond ), 37 °C (down-triangle), 37 °C after preincubation at 60 °C for 30 min (triangle ), 37 °C after preincubation at 66 °C for 30 min (), 60 °C (open circle ), 66 °C (×). Spectra were recorded by following the time course of donor fluorescence (lambda ex = 335 nm, lambda em = 460 nm) from equimolar mixture of alpha -IAEDANS (donor) and alpha -IAF (acceptor). d, effect of preincubation with beta LOW-crystallin at different ratios. alpha -IAEDANS and alpha -IAF were incubated with beta LOW-crystallin at 1:0.5 (diamond ), 1/1 (black-diamond ), and 1:2 (open circle ) w:w: ratios at 60 °C for 30 min. Subunit exchange was carried at 37 °C. Consecutive emission spectra (lambda ex = 335 nm) were collected over 3 h, and the intensities at maximum donor fluorescence (lambda em = 460 nm) were plotted as a function of time.

Subunit Exchange and Chaperone Activity of alpha -Crystallin toward Other Lens Crystallins by FRET-- beta LOW-Crystallin was examined for the effect on alpha -crystallin subunit exchange. IAEDANS- and IAF-labeled alpha -crystallins were heated at 60 °C for 30 min either alone or in the presence of beta LOW-crystallin. All samples were returned to ambient temperature and stored overnight at 4 °C. Subunit exchange experiments were then carried out at 37 °C, and the extent of energy transfer was compared for mixtures containing alpha -crystallins alone or preincubated with beta LOW-crystallin. As can be seen in Fig. 3, a and b, the extent of energy transfer, and therefore the rate of subunit exchange, are significantly lower for alpha -crystallin preincubated with beta LOW-crystallin. The lowest spectra on Fig. 3, a and b, represent the emission spectra (excitation/emission 335/460) after two hours of subunit exchange. The estimated values for the efficiency of transfer (E) for alpha -crystallin alone and for alpha -crystallin preincubated with beta LOW-crystallin are 0.5 and 0.3, respectively. The decrease in the rate of subunit exchange was found proportional to the beta LOW/alpha -crystallin ratio (Fig. 3d). Such a response is in agreement with the formation of beta LOW/alpha -crystallin hetero-complexes and could indicate the presence of multiple binding sites and/or alpha -crystallin association with beta LOW-crystallin oligomers.

Differential Ability of gamma -Family Members to Inhibit alpha -Crystallin Subunit Exchange-- FRET was used to compare the differential ability of the gamma -family members to associate with alpha -crystallin by following the rate of subunit exchange in the presence of bound substrate. Fig. 4b presents the time course of subunit exchange for alpha -crystallin preincubated for half an hour at 66 °C with the different gamma -crystallins at a w:w ratio 1:1. As can be seen, gamma S is the most effective in slowing the alpha -crystallin subunit exchange followed by hgamma Srec, gamma E, gamma A/F, then by gamma D and by gamma B, which are much less efficient. In separate experiments, alpha -crystallin was preincubated at 66 °C with increasing amounts of the different gamma -crystallins. For all gamma -crystallins studied the inhibition of subunit exchange was dose-dependent (Fig. 4, c-f). Ratios of gamma /alpha up to 4:1 (w:w) resulted in corresponding increases of subunit exchange inhibition, suggesting again the presence of multiple binding sites and/or association with gamma -crystallin oligomers. When repeated, on the same day or 1 week later, the subunit exchange experiments consistently provided us with the same results. Moreover, subunit exchange experiments using previously assembled gamma /alpha complexes were reproducible over weeks. It therefore appears that the gamma /alpha -crystallin complexes are particularly stable.


View larger version (37K):
[in this window]
[in a new window]
 
Fig. 4.   alpha -crystallin subunit exchange in the presence of different gamma -crystallins. a, the emission spectra of donor gamma S-IAEDANS (0.05 mg/ml) and acceptor alpha -IAF (0.1 mg/ml) were recorded first at 25 °C using lambda ex = 335 nm (black-diamond ). Then temperature was changed to 66 °C and spectra recorded after 10- (open circle ), 20- (), and 30- (down-triangle) min incubation. Finally, the temperature in cuvette was allowed to return to 25 °C and spectra were recorded again (diamond ). b-g, different gamma -crystallins were compared for their effect on the rate of alpha -crystallin subunit exchange. alpha -IAEDANS and alpha -IAF were preincubated at 66 °C (30 min) with equal volumes of gamma -crystallins solutions or buffer (control). Samples were allowed to return to an ambient temperature before FRET experiments. Equal volumes of preincubated protein solutions were mixed at 37 °C, and the time course for the decrease in donor fluorescence was monitored at lambda em = 460 nm for 60 min. b, lanes from top correspond to alpha  and different gamma  at 1:1 (w:w) ratios for gamma S, gamma Srec, gamma E, gamma A-F, gamma D, gamma B, and alpha  alone. c-g, lanes from top for alpha  and gamma  at different w:w ratios: 1:4 (triangle ), 1:2 (open circle ), 1:1 (diamond ), 1:0.5 (), and 1:0 (black-down-triangle ). c, alpha  and gamma S; d, alpha  and gamma Srec; e, alpha  and gamma E; f, alpha  and gamma D; g, alpha  and gamma B. h, some weak but stable association is recorded at 37 °C between individually heat-modified soluble lens proteins. alpha -IAEDANS, alpha -IAF, and gamma S were individually incubated at 66 °C for 30 min at 0.05 mg/ml each before the return to room temperature. Donor alpha -IAEDANS and acceptor alpha -IAF were preincubated further for 30 min at 37 °C with equal volumes of gamma S (black-down-triangle ) or buffer (black-triangle) and used in subunit exchange experiment as described above. alpha -IAEDANS, alpha -IAF, and beta LOW were treated the same way except for the incubation temperature (60 °C). Subunit exchange was recorded similarly in the presence (down-triangle) or absence (triangle ) of beta LOW.

The Formation of Hetero-complexes at 37 °C Requires "Activated" Partners-- In separate experiments, the subunit exchange assay was used to check whether high temperature modified lens proteins could form hetero-complexes at 37 °C. Proteins at concentrations <= 0.05 mg/ml were heat-treated separately for 30 min at low protein concentrations to avoid precipitation and returned to ambient temperature prior to experiments. All proteins studied remained soluble under these conditions. Subunit exchange was carried out at 37 °C as described in the legend of Fig. 4h. Data showed that there was an inhibition of alpha -crystallin subunit exchange in the presence of beta LOW or of gamma S, therefore confirming the alpha /beta LOW and the alpha /gamma S-crystallin association (Fig. 4h). However, the association was weaker, since the inhibition of subunit exchange by both beta LOW and gamma S was smaller in comparison with data on Fig. 3, b and d. Control experiments showed that the modification by incubation of both alpha - and beta LOW/gamma -crystallin was required for the association to occur. Overall, these data support the hypothesis that the chaperone activity of alpha -crystallin toward the other lens crystallins requires an "activation," which in our case is provided by the temperature induced structural transition, and that this activated state is retained at lower temperature. Reciprocally, only "aggregate competent" beta LOW- or gamma -crystallin can associate to activated alpha -crystallin, and the competent state may be retained at physiological temperatures.

Effect of Bound Substrate on the Fluorescence of Labeled alpha -Crystallin-- Fluorescence characteristics of IAEDANS- and IAF-labeled alpha -crystallin in the presence of different beta LOW- or gamma -crystallins are in support of various binding surfaces in alpha -crystallin. As can be seen from Fig. 5, a-f, there is a differential response in both alpha -IAF and alpha -IAEDANS fluorescence upon binding of beta LOW, gamma S, or gamma D-crystallin. Clearly, the environments around labeled residues were differently affected by binding of each of the substrates, reflecting various binding configurations of alpha -crystallin.


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 5.   Spectral changes in alpha -IAEDANS and alpha -IAF induced on the association with gamma S, gamma D, or beta LOW-crystallins. Spectral characteristics of alpha -IAF (a, c, d) and alpha -IAEDANS (b, d, f) change differently upon temperature-induced association with various lens proteins. alpha -IAF and alpha -IAEDANS were preincubated at 1:0.5 (black-diamond ) or 1:1 (diamond ) w:w ratios for 30 min with equal volumes of beta LOW-crystallin at 60 °C (a, b), gamma S-crystallin at 66 °C (c, d), or gamma D-crystallin at 66 °C (e, f). Spectra of alpha -IAF and alpha -IAEDANS with bound proteins were recorded upon the return to ambient temperature using lambda ex = 495 nm and lambda ex = 335 nm. g, the time course of alpha -IAF fluorescence (lambda ex = 495 nm, lambda em = 512 nm) during the incubation at 66 °C alone () or in the presence of gamma S (1:1, black-square). h, the time course of alpha -IAF fluorescence (lambda ex = 495 nm, lambda em = 512 nm) during the incubation at 60 °C alone (open circle ) or in the presence of beta low (1:1, ).

Time Course of the Associations-- The formation of hetero-complexes requires the activation of both the alpha -crystallin and the beta LOW/gamma substrates. The simultaneous occurrence of these events at high temperatures is illustrated on Fig. 5, g and h. Small aliquots of protein solutions (either alpha  alone or alpha :beta LOW at 1:1, or alpha :gamma S at 1:1 mixtures) were introduced into preheated buffer inside the thermostated cuvette at 60 or 66 °C, and the time course of alpha -IAF fluorescence was recorded immediately after mixing. The upper lanes in Fig. 5, g and h, show the time-dependent increase in alpha -IAF fluorescence at 60 and 66 °C for alpha -crystallin alone, whereas the lower lanes show the alpha -IAF fluorescence in the presence of beta LOW or gamma S. The increase in alpha /IAF fluorescence as a function of time is in agreement with the alpha -crystallin structural changes as mentioned above, whereas the inhibition in the presence of beta LOW/gamma -crystallins seems to indicate a rapid association of substrate with the outside alpha -crystallin subunits. It appears that the binding of beta LOW- or gamma S-crystallin occurs rapidly, since an offset in fluorescence of alpha -IAF starts immediately after the mixing of proteins. All events therefore seem to occur simultaneously. It appears that aggregate competent beta LOW/gamma -crystallin forms rapidly and start to associate with alpha -crystallin at the onset of its structural changes.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

As soon as it was demonstrated in vitro that alpha -crystallin could prevent beta - or gamma -crystallin aggregation, it was hypothesized that such a chaperone activity might be an important factor to preserve in vivo lens transparency under various stress conditions and in aging. The functional model behind was that alpha -crystallin was able to associate beta - or gamma -crystallin at the onset of its denaturation, thus preventing further unfolding and precipitation, possibly protecting against cataract (3, 4, 54). To better evaluate this model, we decided to compare the chaperone properties of native calf alpha -crystallin toward other lens crystallins, beta LOW and gamma , using various techniques, essentially SAXS and FRET but also visible light absorption, gel filtration, gel electrophoresis, Western blotting. The beta LOW- or gamma -crystallin unfolding was induced by heat.

The first point addressed was the structural state of the various partners during the chaperone reaction. It was known that beta LOW- and gamma -crystallin start to unfold at 60 and 66 °C, respectively. From the changes in Trp fluorescence spectra, we have shown that the unfolding remains limited. The beta LOW-crystallin structure seems only slightly more open, whereas changes in individual gamma -crystallin are even fainter. At the low protein concentrations (about 25 µg/ml) used in fluorescence experiments, beta LOW- or gamma -crystallin aggregates after unfolding, as shown by the appearance of a Rayleigh peak, but no insoluble precipitate could be detected. Similar changes could not be followed at high protein concentration, since the conformational changes were immediately followed by crystallin aggregation and precipitation. On the other hand, the data obtained by SAXS at high protein concentrations provided us with quantitative parmeters for the size changes of alpha -crystallin during and after heat treatment. The changes include both a doubling in molecular weight accompanied by the corresponding increase in size and more local changes like the alteration in the environment around Cys residues demonstrated by fluorescence. The changes were found irreversible on return to room temperature and, whenever it was possible to check it, independent of protein concentration. These results extend the previously published observations using other physical techniques like EM (43). They are also consistent with probing studies of recombinant alpha A- and alpha B-crystallin structure with 1,1'-bis(4-anilino)naphthalene-5,5'-disulfonic acid, which indicated an increased exposure of hydrophobic surfaces after heat induced conformational changes (55, 56).

The second point addressed was whether the protection against precipitation was linked to crystallin-crystallin association. This could easily be demonstrated for beta LOW-crystallin, since soluble alpha /beta LOW-crystallin complexes were detected at concentrations up to 20 mg/ml, by both gel filtration and SAXS. For comparison, the detection at high protein concentration of alpha /gamma -crystallin complexes before precipitation could only be demonstrated by Western blotting. At low concentration, however, the presence of alpha /gamma -crystallin complexes was readily demonstrated by FRET between labeled alpha - and gamma -crystallin (Fig. 4a). Overall, the results obtained using SAXS and fluorescence confirmed that the temperature-induced structural modifications were necessary for hetero-complex formation between lens proteins. As a final result it has been shown that separate preincubation at high temperature of alpha -crystallin and of gamma -crystallin may lead, upon mixing at 37 °C, to the formation of alpha /gamma -crystallin complexes. Therefore, the whole of these experiments emphasizes the importance of the structural modifications of both alpha - and beta LOW/gamma -crystallin for the association to take place. On the contrary, the chaperone activity of human recombinant alpha A- or alpha B-crystallin toward other partially unfolded substrates, like insulin or lactalbumin, or thermally denatured alcohol dehydrogenase or citrate synthase, or even gamma D-crystallin, yet denatured by singlet oxygen at 23 °C, does not require the alpha -crystallin structural transition (9, 14, 16, 17, 20, 25, 29, 56, 57).

The third point addressed was the relationship between chaperone properties and subunit exchange. Experiments on subunit exchange are most easily performed by FRET (31, 33, 34). In the present study the probes used were (5-IAF) and (1,5-IAEDANS). We have shown that the rate of subunit exchange measured with these probes and calf alpha -crystallin (which contains both alpha A (75%) and alpha B (25%)) was the same as the one measured previously with other probes and recombinant alpha A (31). The chaperone activity toward other lens proteins occurs, however, at 60 or 66 °C where the rate of exchange is too fast to be measured. A modified subunit exchange assay was designed in which two populations of alpha -crystallin, labeled either with IAF or IAEDANS, are separately incubated at high temperature. The two populations are then brought back to low temperature to stop the reaction, and the rate of exchange is measured at 37 °C by recording the time-dependent decrease in donor fluorescence after mixing of the two populations. The assay demonstrated unambiguously that the temperature-modified alpha -crystallin retains its dynamic organization, i.e. can still exchange subunits at 37 °C. In fact, it exchanges more rapidly (Fig. 3), demonstrating that the temperature-induced structural transition has altered the dynamic structure of alpha -crystallin. The high temperature alpha -crystallin structure therefore appears to be an "activated" structure and the activation to be linked to the dynamic properties. The rate of subunit exchange is not concentration-dependent, which means that subunit exchange most likely occurs through a dissociation mechanism (34). One important implication is that the chaperone effect of alpha -crystallin versus the other lens crystallins can then be evaluated by following the rate of exchange at 37 °C after incubation at 60 or 66 °C for half an hour with the beta LOW- or gamma -crystallin of interest. Fluorescence measurements are done at low protein concentrations and under these conditions, the alpha /beta LOW- and alpha /gamma -crystallin complexes remained soluble in solution and were stable for many days. This allowed us to proceed with the comparison of the different beta LOW- or gamma -crystallins. Such a comparison revealed considerable differences between gamma -crystallins in their ability to interact with alpha -crystallin at high temperature, although gamma -crystallins are homologous two-domain proteins that share from 50% (gamma S with the others) up to 80% (gamma A-F) sequence identity. The more distant gamma S and hgamma Srec were the most effective, with hgamma Srec closely followed by gamma E and gamma A-F, and then by gamma D and gamma B. Despite their close sequence similarity, gamma -crystallins are already known to present different physico-chemical properties, e.g. gamma B unfolds through stable intermediates (C-terminal domain unfolded, N-terminal domain folded), while for gamma S no stable intermediates could be detected under similar conditions (58, 59). Phase separation temperatures are different: around 30 °C for gamma E and gamma A-F, 4 °C for gamma D and gamma B, and no phase separation at all for gamma S, either native or recombinant (60, 61). In the present study, gamma E and gamma A-F that have high phase separation temperatures definitively appear more efficient in forming complexes than the low phase separation temperature gamma D and gamma B. Yet, the differential ability of the different gamma -crystallins to form gamma /alpha -crystallin complexes does not seem to simply follow this well known gamma -crystallin property (62, 63). Interestingly, the most efficient gamma -crystallin is gamma S, either native or recombinant, which is reminiscent of the observation that gamma S appears functionally special within the family. Indeed the gamma S, which in vivo is preferentially localized in the lens cortex, could play a role in protecting against stress (47, 64). Yet the calf gamma S and the hgamma Srec, studied with calf alpha -crystallin as a chaperone, behave in a similar but not identical way. The difference could reflect their different abundance and possibly different specific functions in calf and in human. One may therefore assume that the chaperone assay tests a combination of stability and associative properties of the gamma -crystallins, which could play a role in function. In separate experiments, alpha -crystallin was preincubated with progressively increasing amounts of beta LOW- or gamma -crystallins. All proteins studied were able to interact with alpha -crystallin in a concentration-dependent manner, the inhibition of exchange increasing with increasing substrate-protein ratio and being clearly different for the individual lens crystallins. Such data suggest the presence of multiple binding sites on alpha -crystallin for beta LOW- or gamma -crystallins and/or association with beta LOW- or gamma -crystallin oligomers. Together with the observation that the fluorescence of IAEDANS-labeled alpha -crystallin is affected differently upon binding of the different gamma -crystallins, the results argue for a variety of beta LOW/alpha and gamma /alpha hetero-complexes. The binding stoichiometry could not, however, be determined from such experiments. The whole of the experiments on subunit exchange emphasizes that the dynamic organization of alpha -crystallins is an essential part of the chaperone effect and cannot be dissociated from it. At 60 °C or 66 °C, a few events simultaneously take place: alpha -crystallin increases in size, beta LOW/gamma -crystallins bind to alpha -crystallin to form complexes, beta LOW/gamma -crystallins become aggregate competent. Either the presence of activated alpha -crystallin in the system permits to disrupt the beta LOW/gamma -crystallin aggregates, because of a competition for the binding sites, or the beta LOW/gamma -crystallin association to alpha -crystallin occurs at a faster rate. In any case, the fast subunit exchange process is obviously an essential requirement for structural rearrangement and protein incorporation. It is therefore reasonable to hypothesize, as was done in other studies (65), that subunit exchange is a governing mechanism in providing the necessary rearrangements to accommodate substrate proteins.

Little is known about the structural characteristics of the hetero-complexes. Yet, the inhibition of alpha /IAF fluorescence in the presence of beta LOW/gamma -crystallins would be consistent with substrate coating around alpha -crystallin, rather than insertion between subunits, because such a coating would conserve homotransfer between subunits. Different alpha -crystallin peptides have been identified in recent studies as functional elements in chaperone activity (66, 67). One of these peptides (residues 71-88 of alpha A-crystallin, (22, 68)) overlaps with putative substrate binding site of small heat shock proteins (67). A second putative binding site was allocated to the N-terminal part, involving aromatic residues (67). Recent studies using mutated alpha -crystallin also demonstrated the involvement of the C-terminal extension in its chaperone activity at 37 °C toward insulin (69). However, all binding interfaces that have been proposed were identified at low temperatures (25-37 °C) and may have different properties at the temperatures at which lens heterocomplexes are formed. For instance, according to the small heat shock three-dimensional structures that have been determined (1, 2), one of the proposed binding sites (residues 71-88) of alpha A-crystallin is located on the surface but covered with the C-terminal extension of another monomer. This site could become exposed during subunit exchange via dissociation from oligomer and thus become available for interactions with substrates.

In conclusion, we have shown that a high temperature stress induces a partial unfolding of beta LOW/gamma -crystallins and an irreversible structural transition in alpha -crystallin resulting in a new dynamic structure. The changes effect subunit exchange properties but do not abolish them. In this configuration, alpha -crystallin can form stable soluble complexes with aggregate competent beta LOW- or gamma -crystallins via multiple interactive surfaces over a wide range of protein concentrations. Individual gamma -crystallins can be differentiated by their ability to inhibit subunit exchange. Since the chaperone activity of alpha -crystallin toward other lens proteins required a heat-induced activation in vitro, the biological relevance of such studies was questioned. We therefore wish to emphasize that after our study, it rather appears that the most important requirement for alpha -crystallin to act as a chaperone is that of a dynamic structure. Given the alpha -crystallin structural plasticity, our guess is that, in vivo, alpha -crystallin may be activated in many ways to act as chaperone in a variety of stress conditions and maintain lens transparency.

    FOOTNOTES

* This work was supported by the CNRS, the CNES, and the European Community BIOMED contract "Aging Vision: Crystallins, Visual Acuity, and Cataract."The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ To whom correspondence should be addressed. Tel.: 33-1-44-27-74-51; Fax: 33-1-44-27-37-85; E-mail: Annette.Tardieu@lmcp.jussieu.fr.

Published, JBC Papers in Press, January 31, 2003, DOI 10.1074/jbc.M208157200

    ABBREVIATIONS

The abbreviations used are: FRET, fluorescence resonance energy transfer; IAEDANS, 5-((((2-iodoacetyl)amino)ethyl)amino)naphthalene-1-sulfonic acid; IAF, 5-(iodoacetamido)fluorescein; SAXS, small angle x-ray scattering; EM, electron microscopy.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1. Kim, R., Kim, K. K., Yokota, H., and Kim, S. H. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 9129-9133[Abstract/Free Full Text]
2. van Montfort, R. L., Basha, E., Friedrich, K. L., Slingsby, C., and Vierling, E. (2001) Nat. Struct. Biol. 8, 1025-1030[CrossRef][Medline] [Order article via Infotrieve]
3. Horwitz, J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 10449-10453[Abstract]
4. Wang, K., and Spector, A. (1994) J. Biol. Chem. 269, 13601-13608[Abstract/Free Full Text]
5. Boyle, D., and Takemoto, L. (1994) Exp. Eye Res. 58, 9-15[CrossRef][Medline] [Order article via Infotrieve]
6. Carver, J. A., Guerreiro, N., Nicholls, K. A., and Truscott, R. J. (1995) Biochim. Biophys. Acta 1252, 251-260[Medline] [Order article via Infotrieve]
7. Das, K. P., and Surewicz, W. K. (1995) Biochem. J. 311, 367-370[Medline] [Order article via Infotrieve]
8. Wang, K., and Spector, A. (1995) Invest. Ophthalmol. Vis. Sci. 36, 311-321[Abstract]
9. Andley, U. P., Mathur, S., Griest, T. A., and Petrash, J. M. (1996) J. Biol. Chem. 271, 31973-31980[Abstract/Free Full Text]
10. Borkman, R. F., Knight, G., and Obi, B. (1996) Exp. Eye Res. 62, 141-148[CrossRef][Medline] [Order article via Infotrieve]
11. Arai, H., and Atomi, Y. (1997) Cell Struct. Funct. 22, 539-544[Medline] [Order article via Infotrieve]
12. Blakytny, R., and Harding, J. J. (1997) Exp. Eye Res. 64, 1051-1058[CrossRef][Medline] [Order article via Infotrieve]
13. Raman, B., and Rao, C. M. (1997) J. Biol. Chem. 272, 23559-23564[Abstract/Free Full Text]
14. Horwitz, J., Huang, Q. L., Ding, L., and Bova, M. P. (1998) Methods Enzymol. 290, 365-383[Medline] [Order article via Infotrieve]
15. Muchowski, P. J., and Clark, J. I. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 1004-1009[Abstract/Free Full Text]
16. Rajaraman, K., Raman, B., Ramakrishna, T., and Rao, C. M. (1998) Biochem. Biophys. Res. Commun. 249, 917-921[CrossRef][Medline] [Order article via Infotrieve]
17. Datta, S. A., and Rao, C. M. (1999) J. Biol. Chem. 274, 34773-34778[Abstract/Free Full Text]
18. Muchowski, P. J., Wu, G. J., Liang, J. J., Adman, E. T., and Clark, J. I. (1999) J. Mol. Biol. 289, 397-411[CrossRef][Medline] [Order article via Infotrieve]
19. van Boekel, M. A., de Lange, F., de Grip, W. J., and de Jong, W. W. (1999) Biochim. Biophys. Acta 1434, 114-123[Medline] [Order article via Infotrieve]
20. Abgar, S., Yevlampieva, N., Aerts, T., Vanhoudt, J., and Clauwaert, J. (2000) Biochem. Biophys. Res. Commun. 276, 619-625[CrossRef][Medline] [Order article via Infotrieve]
21. Haley, D. A., Bova, M. P., Huang, Q. L., McHaourab, H. S., and Stewart, P. L. (2000) J. Mol. Biol. 298, 261-272[CrossRef][Medline] [Order article via Infotrieve]
22. Sharma, K. K., Kumar, R. S., Kumar, G. S., and Quinn, P. T. (2000) J. Biol. Chem. 275, 3767-3771[Abstract/Free Full Text]
23. Wang, K., and Spector, A. (2000) Eur. J. Biochem. 267, 4705-4712[Abstract/Free Full Text]
24. Derham, B. K., van Boekel, M. A., Muchowski, P. J., Clark, J. I., Horwitz, J., Hepburne-Scott, H. W., de Jong, W. W., Crabbe, M. J., and Harding, J. J. (2001) Eur. J. Biochem. 268, 713-721[Abstract/Free Full Text]
25. Lindner, R. A., Treweek, T. M., and Carver, J. A. (2001) Biochem. J. 354, 79-87[CrossRef][Medline] [Order article via Infotrieve]
26. Rajaraman, K., Raman, B., Ramakrishna, T., and Rao, C. M. (2001) FEBS Lett. 497, 118-123[CrossRef][Medline] [Order article via Infotrieve]
27. Augusteyn, R. C., Murnane, L., Nicola, A., and Stevens, A. (2002) Clin. Exp. Optom. 85, 83-90[Medline] [Order article via Infotrieve]
28. Bhattacharyya, J., Srinivas, V., and Sharma, K. K. (2002) J. Protein Chem. 21, 65-71[CrossRef][Medline] [Order article via Infotrieve]
29. Carver, J. A., Lindner, R. A., Lyon, C., Canet, D., Hernandez, H., Dobson, C. M., and Redfield, C. (2002) J. Mol. Biol. 318, 815-827[CrossRef][Medline] [Order article via Infotrieve]
30. Reddy, G. B., Narayanan, S., Reddy, P. Y., and Surolia, I. (2002) FEBS Lett. 522, 59-64[CrossRef][Medline] [Order article via Infotrieve]
31. Bova, M. P., Ding, L. L., Horwitz, J., and Fung, B. K. (1997) J. Biol. Chem. 272, 29511-29517[Abstract/Free Full Text]
32. Datta, S. A., and Rao, C. M. (2000) J. Biol. Chem. 275, 41004-41010[Abstract/Free Full Text]
33. Sun, T. X., Akhtar, N. J., and Liang, J. J. (1998) FEBS Lett. 430, 401-404[CrossRef][Medline] [Order article via Infotrieve]
34. Bova, M. P., McHaourab, H. S., Han, Y., and Fung, B. K. (2000) J. Biol. Chem. 275, 1035-1042[Abstract/Free Full Text]
35. Liang, J. J. (2000) FEBS Lett. 484, 98-101[CrossRef][Medline] [Order article via Infotrieve]
36. Bera, S., and Abraham, E. C. (2002) Biochemistry 41, 297-305[CrossRef][Medline] [Order article via Infotrieve]
37. Bova, M. P., Huang, Q., Ding, L., and Horwitz, J. (2002) J. Biol. Chem. 277, 38468-38475[Abstract/Free Full Text]
38. Bours, J. (1996) Ophthalmic Res. 28, 23-31[Medline] [Order article via Infotrieve]
39. Das, K. P., Choo-Smith, L. P., Petrash, J. M., and Surewicz, W. K. (1999) J. Biol. Chem. 274, 33209-33212[Abstract/Free Full Text]
40. Raman, B., Ramakrishna, T., and Rao, C. M. (1995) J. Biol. Chem. 270, 19888-19892[Abstract/Free Full Text]
41. Weinreb, O., van Rijk, A. F., Dovrat, A., and Bloemendal, H. (2000) Invest. Ophthalmol. Vis. Sci. 41, 3893-3897[Abstract/Free Full Text]
42. Burgio, M. R., Kim, C. J., Dow, C. C., and Koretz, J. F. (2000) Biochem. Biophys. Res. Commun. 268, 426-432[CrossRef][Medline] [Order article via Infotrieve]
43. Burgio, M. R., Bennett, P. M., and Koretz, J. F. (2001) Mol. Vis. 7, 228-233[Medline] [Order article via Infotrieve]
44. Abgar, S., Vanhoudt, J., Aerts, T., and Clauwaert, J. (2001) Biophys. J. 80, 1986-1995[Abstract/Free Full Text]
45. Vérétout, F., Delaye, M., and Tardieu, A. (1989) J. Mol. Biol. 205, 713-728[Medline] [Order article via Infotrieve]
46. Vérétout, F., and Tardieu, A. (1989) Eur. Biophys. J. 17, 61-68[Medline] [Order article via Infotrieve]
47. Skouri-Panet, F., Bonnete, F., Prat, K., Bateman, O. A., Lubsen, N. H., and Tardieu, A. (2001) Biophys. Chem. 89, 65-76[CrossRef][Medline] [Order article via Infotrieve]
48. Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve]
49. Guinier, A., and Fournet, G. (1955) Small Angle Scattering of X-rays , Wiley, New York
50. Dubuisson, J. M., Descamps, T., and Vachette, P. (1997) J. Appl. Crystallogr. 30, 49-54[CrossRef]
51. Lakowicz, J. R. (1999) Principles of Fluorescence Spectroscopy , Kluwer Academic/Plenum Publishers, New York
52. Wu, P., and Brand, L. (1994) Anal. Biochem. 218, 1-13[CrossRef][Medline] [Order article via Infotrieve]
53. Tardieu, A., Laporte, D., Licinio, P., Krop, B., and Delaye, M. (1986) J. Mol. Biol. 192, 711-724[Medline] [Order article via Infotrieve]
54. MacRae, T. H. (2000) Cell. Mol. Life Sci. 57, 899-913[Medline] [Order article via Infotrieve]
55. Liang, J. J., Sun, T. X., and Akhtar, N. J. (2000) Mol. Vis. 6, 10-14[Medline] [Order article via Infotrieve]
56. Reddy, G. B., Das, K. P., Petrash, J. M., and Surewicz, W. K. (2000) J. Biol. Chem. 275, 4565-4570[Abstract/Free Full Text]
57. Lindner, R. A., Kapur, A., and Carver, J. A. (1997) J. Biol. Chem. 272, 27722-27729[Abstract/Free Full Text]
58. Wenk, M., Herbst, R., Hoeger, D., Kretschmar, M., Lubsen, N. H., and Jaenicke, R. (2000) Biophys. Chem. 86, 95-108[CrossRef][Medline] [Order article via Infotrieve]
59. Jaenicke, R., and Slingsby, C. (2001) Crit. Rev. Biochem. Mol. Biol. 36, 435-499[Abstract/Free Full Text]
60. Broide, M. L., Berland, C. R., Pande, J., Ogun, O. O., and Benedek, G. B. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 5660-5664[Abstract]
61. Liu, C., Asherie, N., Lomakin, A., Pande, J., Ogun, O., and Benedek, G. B. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 377-382[Abstract/Free Full Text]
62. Lomakin, A., Asherie, N., and Benedek, G. B. (1996) J. Chem. Phys. 104, 1646-1656[CrossRef]
63. Malfois, M., Bonneté, F., Belloni, L., and Tardieu, A. (1996) J. Chem. Phys. 105, 3290-3300[CrossRef]
64. Sinha, D., Wyatt, M. K., Sarra, R., Jaworski, C., Slingsby, C., Thaung, C., Pannell, L., Robison, W. G., Favor, J., Lyon, M., and Wistow, G. (2001) J. Biol. Chem. 276, 9308-9315[Abstract/Free Full Text]
65. van Montfort, R., Slingsby, C., and Vierling, E. (2001) Adv. Protein Chem. 59, 105-156[Medline] [Order article via Infotrieve]
66. Sharma, K. K., Kaur, H., and Kester, K. (1997) Biochem. Biophys. Res. Commun. 239, 217-222[CrossRef][Medline] [Order article via Infotrieve]
67. Sharma, K. K., Kumar, G. S., Murphy, A. S., and Kester, K. (1998) J. Biol. Chem. 273, 15474-15478[Abstract/Free Full Text]
68. Sreelakshmi, Y., and Sharma, K. K. (2001) J. Protein Chem. 20, 123-130[CrossRef][Medline] [Order article via Infotrieve]
69. Pasta, S. Y., Bakthisaran, R., Tangirala, R., and Mohan Rao, C. (2002) J. Biol. Chem. 277, 45821-45828[Abstract/Free Full Text]


Copyright © 2003 by The American Society for Biochemistry and Molecular Biology, Inc.