Preliminary Studies on the Aggregation Process of Alpha-Crystallin

Exp. Eye Res. (1997) 65, 255–266

Preliminary Studies on the Aggregation Process of Alpha-Crystallin E L L E N W. D O S S*, K A T H L E E N A. W A R D    J A N E F. K O R E T Z Center for Biochemistry and Biophysics, Department of Biology, Rensselaer Polytechnic Institute, Troy, New York 12180-3590, U.S.A. (Received Rochester 22 October 1996 and accepted in revised form 30 March 1997) The mechanism by which α-crystallin subunits form the native 800 kD aggregate is currently unknown. Experiments were performed to investigate the mechanism for this process. Gel-filtration Fast Performance Liquid Chromatography (FPLC) and Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDSPAGE), with and without cross-linking with glutaraldehyde, indicate that α-crystallin undergoes a concentration-dependent aggregation process. The denaturation of α-crystallin, and its subsequent renaturation and reaggregation, lead to the formation of several different species. At very low concentrations (! 0±5 µ), only monomeric and}or dimeric species exist. With a ten-fold increase in αcrystallin concentration from 0±05 µ to 0±5 µ, the amount of the monomeric}dimeric species increases to a plateau coincident with the appearance of a tetrameric species at 0±5 µ. With an additional tenfold increase in concentration from 0±5 µ to 5 µ, the amount of the tetrameric species increases and levels off to its own plateau coincident with the appearance of the native 800 kD α-crystallin aggregate at 5 µ. The amount of the native species is extremely small at this concentration, but increases sharply and linearly with increasing concentration, while the concentrations of monomeric}dimeric and tetrameric species remain constant. The concentration at which the relative amount of the native species begins to increase sharply is within the range of the critical micelle concentration previously characterized. # 1997 Academic Press Limited Key words : α-crystallin ; aggregation ; micelles ; denaturation}renaturation ; gel filtration fast performance liquid chromatography ; sodium dodecyl sulfate polyacrylamide gel electrophoresis ; crosslinking.

1. Introduction α-crystallin, the major protein of the mammalian lens, exists as an assembly or assemblies of two different subunits, αA-crystallin (αA) and αB-crystallin (αB). αA and αB are separate gene products, with molecular masses of approximately 20 kD (Hoenders and Bloemendal, 1981). In humans, αA and αB coexist in the lens at a molar ratio of 3αA : 1αB. This ratio, however, can vary among species. αA and αB can be separated from each other by isoelectric focusing because the pI of the two differ ; αA is acidic (pI ¯ 5±4–5±9) and αB is basic (pI ¯ 6±6–7±4) (Van der Ouderaa, de Jong and Bloemendal, 1973 ; Van der Ouderaa et al., 1974 ; Schoenmakers and Bloemendal, 1968). αA2, the major α-crystallin polypeptide chain, and αB2, are the primary gene products, while αA1 and αB1 are produced from these polypeptides through post-translational phosphorylation. In the lens, these phosphorylated forms represent 10 % of each gene product (Van der Ouderaa et al., 1973 ; Voorter et al., 1986 ; Chiesa et al., 1987a, 1987b ; de Jong, 1981). In addition to these four isoforms, degradation products and modifications may arise with aging or maturation by a variety of pathways (Hoenders and Bloemendal, 1981). * Corresponding author.

0014-4835}97}080255­12 $25.00}0}ey970337

The mammalian lens crystallins are related to proteins that have non-refractive functions. αCrystallin belongs to a family of small heat shock proteins, and, β-crystallin and γ-crystallin belong to a superfamily of microbial stress proteins (Ingolia and Craig, 1982 ; Wistow, 1990 ; Crabbe and Goode, 1994). α-crystallin has been found in a number of specialized non-lenticular tissues and organs (Moscona et al., 1985 ; Lewis et al., 1988 ; Iwaki et al., 1989), where it may serve as a molecular chaperone (Horwitz, 1992). αB-crystallin has been found in the heart, lung, spinal cord, brain, kidney, skin, muscle, and retina, while αA-crystallin has been identified in the spleen and thymus (Bhat and Nagineni, 1989 ; Kato et al., 1991 ; Srinvasan, Nagineni and Bhat, 1992). The lens is the only tissue identified in which both α-crystallin gene products are expressed. In addition to providing a refractive function in the lens, α-crystallin may serve as a molecular chaperone, protecting other lenticular crystallins and proteins from denaturation and}or aggregation, which would otherwise eventually lead to glare effects and opacification. When α-crystallin is isolated from the lens at room temperature by gel filtration chromatography, aggregates of 30–50 subunits are resolved. These aggregates have an average apparent molecular mass of approximately 800 kD. From many experiments performed, α-crystallin aggregates appear to exist as a het# 1997 Academic Press Limited

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erogeneous population of roughly spherical particles (Siezen and Hoenders, 1979 ; Koretz and Augusteyn, 1988 ; Augusteyn, Koretz and Schurtenberger, 1989). Hoenders, Siezen and Bindels have proposed a concentric three-layered model as a representation for αcrystallin (Bindels, Siezen and Hoenders, 1979 ; Siezen, Bindels and Hoenders, 1980). In this model, as well as in some variations of this model (e.g., Tardieu et al., 1986), native α-crystallin is represented as three concentric layers of subunits, with well-defined locations and specific molar ratios of the isoforms, αA and αB. Variations in molecular mass and shape among α-crystallin particles are explained by different occupancy levels in the outer layer of subunits. The concentric model is incompatible with observations indicating equivalent microenvironments for all subunits (Augusteyn and Koretz, 1987 ; Carver, Aquilina and Truscott, 1993 ; Augusteyn, Ghiggino and Putilina, 1993). Two other models, both of which place α-crystallin subunits in equivalent microenvironments, have been proposed more recently. Carver, Aquilina and Truscott (1994) have hypothesized that α-crystallin may aggregate into a double-ring structure, similar to that exhibited by GroEl (Carrascosa et al., 1990) and thermosome (Phipps et al., 1993). While this model provides identical microenvironments for subunits, it does not explain completely variations in aggregate size and molecular weight. Furthermore, no particle with a structure consistent with this model has ever been observed by electron microscopy. Augusteyn and Koretz (1987) have suggested that the structure of αcrystallin is best explained by a micellar model, in which the amphipathic organization of the subunits leads to a non-unique structure held together by hydrophobic interactions. Several sets of experiments, designed specifically to test this model (e.g., Radlick and Koretz, 1992), gave results consistent with micelle-like behavior and provided estimates of the critical micelle concentration (CMC) for α-crystallin (0±18–0±25 m, or 3±5–5±0 mg ml−"). Since, however, a number of laboratories routinely work with αcrystallin at concentrations well below the minimum for the CMC range, and can show that it is in the ‘ native ’ state, the micellar model for α-crystallin appears unacceptable as well, unless it can be shown that the aggregate is kinetically ‘ trapped ’. An increased understanding of the actual mechanism of α-crystallin association would not only shed light on the properties of the subunit, but might also provide information about the structure and stability of the aggregate. However, obtaining this information requires an approach quite different from that used for the study of other aggregating proteins. In other systems, the monomer or subunit can be solubilized, and the transition from subunit to aggregate followed as a function of initial protein concentration. Because α-crystallin aggregates apparently cannot be solubilized in aqueous medium, except in the presence of

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detergents (Horwitz, personal communication), the experiments described herein utilize denatured αcrystallin in 9  urea as the starting point of the renaturation reaction. The denatured protein concentration is varied, and renaturation and reaggregation are allowed to occur. Characterization of the resultant aggregate populations, as a function of protein concentration, by gel filtration FPLC and other techniques, indicates that (a) α-crystallin undergoes a multi-step aggregation process ; (b) the 800 kD aggregate does not appear as a major part of the populations until the initial protein concentration approaches the CMC previously estimated for αcrystallin ; and, (c) the intermediate aggregation species are in equilibrium with the 800 kD species at and above the CMC. Since solutions of α-crystallin at the same concentrations before and after the denaturation}renaturation process have very different population characteristics, it is suggested that the 800 kD α-crystallin aggregates are kinetically trapped. 2. Materials and Methods Preparation of α-Crystallin Bovine calf lenses, extracted from animals less than three months old, were obtained from Falls Packing Company (Oriskany Falls, NY, U.S.A.) and stored at ®50°C until use. α-crystallin was prepared using a modification of the method of Thompson and Augusteyn (1983). Thawed lenses were handhomogenized in buffer (0±1  NaCl (Sigma), 50 m imidazole (Sigma), 0±02 % NaN (Sigma), pH 7±5), and $ centrifuged at 18 000 g, at 4°C for 30 minutes (Sorvall RC-5B Refrigerated Superspeed Centrifuge, SS-34 Rotor). The supernatant was placed on a Sepharose CL-6B column (Pharmacia), which had been equilibrated with the homogenizing buffer. Fractions were collected in 3±7 ml volumes ; and, those fractions corresponding to the α-crystallin peak were pooled, concentrated to 0±5 m in an Amicon ultrafiltration cell (Model 8050) using a YM5 membrane, and stored at 4°C until use. Preparation of Denatured Samples of α-Crystallin α-Crystallin concentrations were determined spectrophotometrically by measuring the absorbance at 280 nm. A correction for any turbidity was determined by measuring the absorbance at 360 nm. The absorbance at 360 nm was subtracted from the absorbance at 280 nm and, the resulting absorbance was divided by the extinction coefficient of 0±83. Concentration determined using the Bio-Rad Protein Assay Kit, based on the method of Bradford, agreed well with those measured spectrophotometrically. αcrystallin, at a concentration of 0±5 m, was diluted with homogenizing buffer (0±1  NaCl, 50 m imidazole, 0±02 % NaN , pH 7±5) to obtain a series of $ samples ranging in concentration from about 0±1 m

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to 0±25 µ. These samples were placed in washed and deionized Spectra}Por 3 molecularporous membrane tubing (MWCO : 3500 ; Spectrum Medical Industries, Houston, TX, U.S.A.), sealed with color-coded Spectra} Por clips. Samples were dialysed against 9  urea (Sigma) for 24 hr to ensure complete denaturation. The 9  urea had been deionized using analytical mixed bed resin AG 501-X8 (D) 20–50 mesh (BioRad). Bagged and coded samples were transferred to homogenizing buffer and renatured for 24 hr. The dialysis medium was changed every half hour for the first three hours, every hour for the next eight hours, and at least once during the next thirteen hours. The final concentrations of the samples were determined spectrophotometrically at 278 nm. In addition, the absorbance at 360 nm was measured for each of the samples. This measurement of turbidity was used to correct the final concentrations by subtracting the absorbance measured at 360 nm from that measured at 278 nm. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE), using the method of Laemmli (1970), was used to show that the native 3 : 1 ratio of αA : αB was conserved in renatured samples. Isoelectric focusing, using both native and denaturing conditions, examined samples before and after renaturation to show that no significant carbamylation of α-crystallin occurred in the presence of urea. To compare the functionality of the native and renatured samples, samples were evaluated for their molecular chaperone-like activity before and after urea treatment (Horwitz, 1992). Molar ratios of 20 : 1, 15 : 1, 10 : 1, and 5 : 1 γ : α-crystallin aggregate were used to test the ability of native and renatured αcrystallin to protect γ-crystallin against heat denaturation. γ-crystallin was obtained during the initial purification from lenses. Samples were heated to 66°C and the relative scattering determined by absorbance readings at 360 nm over 50 minutes. Heated and unheated control tests were also performed for molar ratios of γ : α-crystallin of 1 : 0. Separation and Characterization of Protein Species with Gel Filtration Chromatography by Fast Performance Liquid Chromatography (FPLC)

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F. 1. Aggregation of γ-crystallin at 66°C in the presence and absence of α-crystallin, native and denatured} renatured. In each experiment, 0±1 mg of γ-crystallin was used with the following additions : (A) Curve 1 (E), no αcrystallin ; 2, (+) 0±2 mg native α-crystallin ; 3, (^) 0±2 mg denatured}renatured α-crystallin ; (B) Curve 1, (E) no α-

After renaturation, 100 µl of each α-crystallin sample was separated by Fast Performance Liquid Chromatography, using Superose 6 HR 10}30 and Superdex 75 HR 10}30 FPLC columns (Pharmacia), with optimal molecular weight ranges of 5 kD to 5000 kD and 3 kD to 70 kD, respectively. Samples

crystallin ; 2, (+) 0±267 mg native α-crystallin ; 3, (^) 0±267 mg denatured}renatured α-crystallin ; (C) Curve 1, (E) no α-crystallin ; 2, (+) 0±4 mg native α-crystallin ; 3, (^) 0±4 mg denatured}renatured α-crystallin ; (D) Curve 1, (E) no α-crystallin ; 2, (+) 0±8 mg native α-crystallin ; 3, (^) 0±8 mg denatured}renatured α-crystallin.

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were eluted at pressures of 162 psi and 144 psi, respectively, for 60 minutes using a Waters 650 Advanced Protein Purification System (Waters). Homogenizing buffer (0±1  NaCl, 50 m imidazole, 0±02 % NaN , pH 7±5) was used as the mobile phase. The $ elution profile was monitored at 278 nm with a Lambda-Max Model 481 LC Spectrophotometer and stored for later display and analysis using a Maxima 820 Chromatography Workstation. Sieving was complete at approximately t ¯ 45 minutes. A molecular weight calibration curve for each column was devised using the Gel Filtration Molecular Weight Markers kit MW-GF-1000 and MW-GF-70 (Sigma). Further calibrations were performed using both α-crystallin and bovine serum albumin (Sigma) at known concentrations to determine the relationship between protein concentration and the area under the elution peaks for each column type. Separation and Characterization of Protein Species by Crosslinking with Glutaraldehyde and by SDS-PAGE Gel Electrophoresis Two samples of α-crystallin at concentrations of 0±05 m and 0±025 m were crosslinked with 0±25 % glutaraldehyde (v}v) (Sigma) at 4°C. The reaction was quenched by the addition of 1  NH Cl (Sigma) at 5, % 10, 15, 20, 30, 45, and 60 minutes. The samples were then dialysed against homogenizing buffer (0±1  NaCl, 50 m imidazole, 0±02 % NaN , pH 7±5) for 1 hr, $ separated by SDS-PAGE Gel Electrophoresis using a 12 % gel (Laemmli, 1970), and visualized by Coomassie Brilliant Blue Stain (Sigma) followed by Silver Stain (Sigma). 3. Results Functional Characterization of Renatured α-Crystallin To test if renatured samples of α-crystallin retained functional characteristics similar to native α-crystallin, the molecular chaperone-like activity of renatured samples was compared to that of native samples of αcrystallin, using the protocol of Horwitz (1992). γcrystallin was combined with α-crystallin, native or renatured, at molar ratios of 20 : 1, 15 : 1, 10 : 1, and 5 : 1 γ : α aggregate. Controls were performed at a ratio of γ : α of 1 : 0, heated and unheated. The samples were heated for approximately 45 minutes and A nm of $'! the samples was measured during this time interval with a Beckman DU-2 Spectrophotometer at 0, 6, 12, 18, 24, 30, 36, and 42 minutes. The renatured αcrystallin samples were found to have the same ability as native α-crystallin to prevent the heat-induced denaturation of γ-crystallin [Fig. 1(A)–(D)] from a 20 : 1 to a 5 : 1 molar ratio of γ : α aggregate. These results indicate that α-crystallin recovered following denaturation in 9  urea and subsequent renaturation was indeed a functional species. This test was performed in light of the fact that α-crystallin,

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denatured in guanidine hydrochloride and subsequently renatured into homogenizing buffer, exhibits a severely reduced ability to provide a chaperone-like protection to γ-crystallin (Koretz, Doss and Reid, 1997). In addition, van Boekel et al. (1996) demonstrated that when the two isoforms of α-crystallin are purified and their phosphorylated and non-phosphorylated forms separated by anion exchange chromatography, the degree of chaperone-like activity is diminished for all forms except αA1, the phosphorylated αA-crystallin isoform. Characterization of α-Crystallin Populations Using Gel Filtration Chromatography by FPLC α-crystallin, when eluted through the Superose 6 HR 10}30 FPLC column (optimal globular molecular weight range of 5–5000 kD) after purification, exhibits a single broad peak at approximately 25 minutes (Fig. 2). This profile is obtained regardless of the time which has elapsed between purification and gel filtration. The peak is present at concentrations ranging from 0±5 µ to 0±5 m, and the area under the curve increases in direct proportion with concentration to the limit of the UV detector (25 µ). Based on the calibration curve for the column, the molecular weight of this species is approximately 800 kD. This is the ‘ native ’ α-crystallin aggregate. When the same concentration range is examined after urea denaturation and renaturation, a variety of elution peaks are observed, depending on sample protein concentration (Fig. 3). At the very lowest concentrations (0±25 µ–0±5 µ), a doublet peak is observed at about 45–46 minutes (Fig. 3, Curve a), with an apparent molecular weight of 18 kD. This doublet increases in size with increasing concentration, but levels off to a constant area upon the appearance of a second, single peak eluting at about 38–40 minutes. This 38–40 minute peak appears at a concentration of approximately 0±5 µ (Fig. 3, Curve b) ; the apparent molecular weight of this species is approximately 45–60 kD. With increasing concentration, the 38–40 minute peak also increases in average area, but begins to level off at about 0±05 m. At a concentration of 5±0 µ, an extremely small amount of a third species appears at approximately 25 minutes (Fig. 3, Curve c). This species has an elution time and molecular weight very similar to that of the ‘ native ’ particle. As the area of the species with an apparent molecular weight of 45–60 kD begins to level off between 0±05 m and 0±1 m (which is within the broad range of critical micelle concentration for α-crystallin), the amount of this ‘ nativesized ’ particle begins to increase rapidly and continues to do so over the remainder of the concentration range sampled. In order to characterize the low molecular weight species more accurately, the experiments were repeated over the same concentration range using a

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F. 2. Typical elution profile of the native α-crystallin aggregate from gel filtration chromatography, using Superose 6 HR 10}30 FPLC column, of a native sample of α-crystallin monitored at 278 nm. Y-axis represents relative absorbance at 280 nm.

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F. 3. Elution profiles from Superose 6 HR 10}30 FPLC column of denatured}renatured samples of α-crystallin at various concentrations. (A) Concentration of α-crystallin : 0±25 µ (0±005 mg ml−"). (B) Concentration of α-crystallin : 5±4 µ (0±108 mg ml−"). (C) Concentration of α-crystallin : 47 µ (0±937 mg ml−"). Note appearance of 35 minute species, followed by the appearance of the 24 minute species with increasing concentration. Y-axis represents relative absorbance at 280 nm. Chromatograms are stacked to facilitate comparison.

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F. 4. Elution profiles from Superdex 75 HR 10}30 FPLC column of denatured}renatured samples of α-crystallin at various concentrations. (A) Concentration of α-crystallin : 0±25 µ (0±005 mg ml−"). (B) Concentration of α-crystallin : 1±05 µ (0±021 mg ml−"). (C) Concentration of α-crystallin : 4±3 µ (0±086 mg ml−"). Note the appearance of the 16 minute species with increasing concentration. Y-axis represents relative absorbance at 280 nm. Chromatograms are stacked to facilitate comparison.

Superdex 75 HR 10}30 FPLC column, with an optimal globular protein separation range of 3–70 kD. At the very lowest concentrations, 0±25 µ to 0±5 µ, two peaks are observed at about 42 and 45 minutes, with apparent molecular weights of ! 10 kD (Fig. 4, Curve a). These two peaks increase in area with an increase in concentration from 0±25 µ to 0±5 µ, but level off to a constant area at a concentration of 0±5 µ. This increase in concentration results in the appearance of a protein species, eluting at 16 minutes, with an apparent molecular weight of 70–80 kD (Fig. 4, Curve b). The area under the peak for this species increases with increasing concentration (Fig. 4, Curve c) and levels off approximately at 0±1 m α-crystallin. The presence of species eluting from the Superdex 75 HR 10}30 column with apparent molecular weights below that of either of the α-crystallin monomers suggests either that a portion of the protein sample is severely degraded or that the monomeric form of α-crystallin is significantly asymmetric, since estimated molecular weights from this technique are based on the assumption that the eluted proteins possess a roughly spherical shape. To investigate these questions, two sets of experiments were performed. In the first set, renatured α-crystallin samples at concentrations of 0±05 m and 0±025 m were cross-

linked with 0±25 % glutaraldehyde (v}v), and their mobility on 12 % SDS-PAGE gels compared to that of non-cross-linked samples. Controls were performed with and without the addition of α-crystallin to the reaction mixture of glutaraldehyde and reaction quencher, NH Cl. Three separate species were clearly % visible in each cross-linked sample, corresponding to molecular weights of 65–75 kD (tetrameric species), 35–45 (dimeric species) and 18–20 kD (monomeric species) (Fig. 5) ; additional species of higher molecular weight were also seen, but in much smaller amounts. No band with a molecular weight lower than that of intact α-crystallin monomers was observed, even after silver staining, indicating that the protein was not severely degraded. There was also material which did not enter the gel. This species is presumably the 800 kD ‘ native ’ aggregate of α-crystallin. The apparent molecular weights of the crosslinked species agreed well with the apparent molecular weights observed from FPLC gel filtration chromatography. For the non-cross-linked controls, bands were observed at the same mobility as, and reflected the native 3 : 1 ratio of, αA- and αB-crystallin subunits. In the second set of experiments, samples from each of the Superdex 75 HR 10}30 FPLC column elution peaks were cross-linked with 0±25 % glutaraldehyde

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F. 5. 12% SDS-PAGE of cross-linked and non-cross-linked denatured}renatured α-crystallin samples. Lane 1 : blank ; Lanes 2–5 : denatured}renatured sample of α-crystallin at various time points, 2, 5, 15, 30 minutes, respectively, in crosslinking ; Lane 6 : non-crosslinked denatured}renatured α-crystallin sample ; Lane 7 : SDS-PAGE molecular weight markers (Sigma). Lane 8 : native α-crystallin standard ; Lanes 9–10 : blanks. Lanes 2–5 demonstrate the three different species involved in the aggregation process. Degree of crosslinking does not increase with time as the reactions were fully quenched. Note the 3 : 1 ratio of αA : αB seen in both the denatured}renatured and native α-crystallin samples.

(v}v). Their mobility, on 12 % SDS-PAGE gels, was compared to that of non-cross-linked aliquots from the same peaks. Three broad bands were seen in the 16 minute peak sample, corresponding to molecular masses of about 70–80, 30–40 and 18–19 kD. Again, these molecular weights correspond well with the monomeric}dimeric and tetrameric species observed by gel filtration chromatography. No bands were seen in the cross-linked samples corresponding to the 42 minute and 45 minute peaks, even after Silver Stain, since the concentration of protein in these samples was extremely low after dilution during crosslinking. In the control experiment, only bands with the same mobility as those of monomeric αA- and αB-crystallin were seen, and the 3αA : 1αB molar ratio between the two isoforms appeared to be preserved.

Turbidity of Renatured Samples : The Monomer}Dimer Transition After the denaturation}renaturation process, it was observed that samples of α-crystallin at concentrations between 0±05 µ and 15 µ (0±001 mg ml−") and 0±3 mg ml−") seemed to be extremely turbid, as determined by a significant increase in the absorbances measured at 278 and 360 nm. Correction for turbidity using the standard method, moreover, resulted in concentration estimates much higher than the initial concentrations for samples within this range. In contrast, samples at higher concentrations exhibited absorbance readings at 278 nm, before and

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after denaturation and renaturation, quite close to that of the initial concentration estimate, using the standard correction method. The standard method subtracts the Abs nm from the Abs nm to $'! #() minimize the contribution of turbidity to the Abs nm, the wavelength which allows for measure#() ment of protein concentration. To investigate this phenomenon further, the factor necessary to correct for turbidity was determined several times for a range of concentrations, averaged, and plotted as a function of concentration [Fig. 6(A)]. For denatured}renatured samples in the low initial concentration range previously mentioned, a much higher factor was required, on average, to correct for the increase in turbidity of these samples. The factor necessary to correct the concentration of samples decreases in magnitude as the initial α-crystallin concentration increases. The concentration range over which this empirical correction is required is precisely that range where only the monomeric}dimeric species is observed [Fig. 6(B)]. Curve 1 represents the relative amount of the monomeric}dimeric species over a portion of the concentration range examined. Curve 2 represents a scaled correction factor over this same concentration range. The monomeric}dimeric species is present in all renatured samples. The turbidity, however, begins to decrease at approximately the concentration at which the tetrameric species constitutes the major αcrystallin species and the native species first appears. It is suggested that the excess turbidity is due to the relatively insoluble monomer of α-crystallin. If this is the case, then the turbidity of the monomeric solution would decrease with increasing concentration upon the formation of a dimeric or tetrameric species, which would shield the hydrophobic portion of the subunit more effectively against interaction with the aqueous medium.

A Possible Aggregation Scheme for α-Crystallin Having characterized the apparent molecular weight for each of the FPLC gel filtration species, and having shown that none of the samples is contaminated with severely degraded α-crystallin subunits, it is now possible to characterize the appearance and relative amounts of these species as a function of increasing protein concentration. At solutions well below micromolar, the only species present have apparent molecular weights of 18 kD, using the Superose 6 HR 10}30 FPLC column, and slightly less than 10 kD, using the Superdex 75 HR 10}30 FPLC column. This is either a significantly asymmetric monomer, an asymmetric dimer, or a combination of both. Since a doublet peak is seen, it is possible either that the column is separating monomeric αA and αB or that it is separating monomers and dimers. It is impossible at this point to distinguish between these two possibilities.

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F. 6. Relationship between initial concentration and turbidity of denatured}renatured α-crystallin samples. (A) (E) Curve demonstrates that as the initial concentration of denatured}renatured samples of α-crystallin increases, the factor necessary to correct for turbidity in the samples decreases in magnitude. Point of inflection of this curve occurs at approximately 5 µ (0±1 mg ml−"). At this concentration of α-crystallin, the native 800 kD aggregate begins to form. (B) Curve 1, (*) Relative amount of α-crystallin monomeric}dimeric species over a given α-crystallin concentration range as determined by gel filtration fast performance liquid chromatography (FPLC) followed by peak area integration analysis ; Curve 2, (U) Factor necessary to correct for increase in turbidity of 9M denatured}renatured α-crystallin samples. Correction factor (U) is scaled by 1¬104 to allow for direct comparison of curves 1 (*) and 2 (U).

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F. 7. Summary of concentration dependent aggregation process of α-crystallin. Aggregation process over entire concentration range studied (0–0±25 m, 0–5 mg ml−" αcrystallin). Curve 1, (+) 800 kD native α-crystallin particle ; 2, (E) 70–80 kD tetrameric species ; 3, (_) monomeric} dimeric species.

With increasing concentration, the amount of the monomeric}dimeric species increases, reaching a concentration plateau, but continuing to be present at

this level, with the appearance of a species at about 0±5 µ. This species is probably a tetramer, since its molecular weight is approximately the same by both FPLC (Superdex 75 HR 10}30 column : 70–80 kD) and, in cross-linked form, by SDS-PAGE (65–80 kD). This species increases in amount with increasing solution concentration, leveling off to its own plateau level with the appearance of the 800 kD aggregate, whose size}molecular weight}mobility, as determined by FPLC, is comparable to that of the native species. The native aggregate is first observed, in a very small amount, at about 5 µ. Thus, for each transition to a larger species, a concentration increase of approximately a factor of 10 is required. It should be noted as well that, at and above the concentration required for the appearance of each new species, the smaller species continue to coexist at their plateau levels. These results are summarized in Fig. 7 and a simple mechanism for α-crystallin aggregation can be derived from it : [monomer % dimer] % tetramer U ‘ native ’ aggregate [20 % 40 kD] % 65–80 kD U 800 kD

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4. Discussion The results obtained in this study indicate that αcrystallin, like other aggregating proteins, undergoes a concentration-dependent aggregation process. At very low concentrations, α-crystallin is in monomeric, then dimeric form. The native aggregate size and molecular weight are approached at protein concentrations roughly three orders of magnitude higher than that at which monomers}dimers are initially detectable. Recently, Dudich et al. (1995) have shown, using difference adiabatic scanning microcalorimetry (DASM) and circular dichroism (CD), that small heat shock proteins (Hsp25, Hsp27) have a melting pattern and secondary, tertiary, and quaternary structures similar to α-crystallin. This is in agreement with the revealed homology of primary structure of these proteins. In addition, according to DASM, the minimum cooperative melting structure is the Hsp25}27dimer (Dudich et al., 1995). The fact that the minimum cooperative melting structure for the heat shock protein is a dimer corresponds well with the findings presented here. In addition, Kanterow et al. (1995) showed that αA-crystallin and αB-crystallin tetramers (80 kD) could be formed by the addition of 1 % deoxycholate. The data presented by Kanterow et al. (1995) demonstrate that a structural requirement for enhanced α-crystallin autophosphorylation is a tetrameric species. These data, and the data presented in this communication, support the fact that a tetrameric species seems to be a fundamental intermediate aggregate in the process which leads to a functionally and structurally competent 800 kD aggregate. Dissocation}reassociation experiments have been performed by a number of different investigators (Siezen, Bindels and Hoenders, 1978 ; Siezen and Bindels (1982) ; Siezen and Owens, 1983 ; Tardieu et al., 1986). The experimental protocols followed in the present communication are different from those employed by Siezen et al. (1978) and Tardieu et al. (1986). This may explain why varying molecular weights for the reassociated α-crystallin have been obtained by different groups. Experimental results by Raman, Ramakrishna and Rao (1995) and those presented in this communication, determined that the molecular weight of the reassociated α-crystallin aggregate closely approached the molecular weight of the native aggregate (800 kD). Tardieu et al. (1986). Siezen et al. (1978) ; (Siezen and Bindels, 1982), and Siezen and Owens (1983) found the molecular weight of the reassociated aggregate to be considerably smaller than that of the native aggregate. Siezen et al. (1978) denatured α-crystallin by directly adding urea to an α-crystallin solution to attain an appropriate concentration of 6  urea (0±04 % DTT) and allowing incubation for 2 hr at room temperature. Urea was removed by dialysis in the cold room for 24 hr. Tardieu et al. (1986) dissociated α-crystallin in 40 m

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phosphate (pH 6±8, 0±01 % NaN , 1 m EDTA, 0±2 m $ DTT) supplemented with 6  urea for 24 hr. Reassociation was carried out by dialysing out urea for 24 or more hours with many buffer changes. The method of denaturation (dialysis vs. direct addition), time course for denaturation (2 hr vs. 24 hr), concentration of denaturant (6  urea vs. 9  urea), conditions for renaturation (cold room vs. room temperature ; number of buffer changes during 24 hr renaturation period), diameter of dialysis tubing used for denaturation}renaturation dialysis, concentration of α-crystallin used during experiments, and analysis method (HPLC vs. FPLC), are factors that may affect the results of what seem to be similar experiments. Unlike many other aggregating systems, α-crystallin exists at intermediate concentrations in the form of aggregated intermediates. It does not exhibit seed formation or a critical concentration, below which only monomers are observed, and above which, the mature aggregate structure is observed in equilibrium with monomers. Instead, at and above the minimum protein concentration necessary for ‘ native ’ aggregate formation, the native aggregate is in equilibrium with all of the smaller species, each of which is present at a characteristic concentration. It is also important to note that the concentration at which the amount of 800 kD aggregates increases sharply (2 mg ml−"), relative to other species of α-crystallin, approaches the critical micelle concentration range previously characterized (Radlick and Koretz, 1992). It is possible, based on these studies, that the same combination of α-crystallin species may exist in vivo. Of course, at the very high concentration of αcrystallin present in the lens, the percentage of protein in the smaller forms is negligible, and would not appear in the same gel filtration column fractions as the 800 kD species. The ‘ native ’ particle itself appears to be quite stable once it has formed. Neither storage over time nor dilution well below that of the critical micelle concentration leads to the appearance of these smaller species. Theoretically, dilution of a solution of 800 kD particles below the concentration needed for their formation should lead to the appearance of these other species and the disappearance of the 800 kD aggregate, in a reversal of the experiments performed herein. Since, however, the denaturation of αcrystallin and its subsequent renaturation and reaggregation leads to the formation of a variety of species, rather than a single 800 kD particle, it is possible that the native structure, once formed, is kinetically ‘ trapped ’. Since neither storage over time nor dilution to a concentration well below that of the critical micelle concentration results in the dissociation of the native particle, the dissociation of the native α-crystallin particle is concluded to be extremely slow. Most likely, the probability of this reverse reaction approaches zero. By unfolding α-crystallin and allowing the protein to refold, it is possible to examine the

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requirements necessary for shielding the hydrophobic regions of α-crystallin subunits from aqueous solvent. It is assumed that the native 800 kD aggregate of αcrystallin is the lowest free energy structure, since an input of energy or change in environment is necessary for dissociation of the native aggregate. For example, the addition of heat alters the structure of α-crystallin by exposing hydrophobic surfaces which may lead to an enhanced ability of the aggregate to bind to and provide a chaperone-like protection for other proteins which may experience heat-induced aggregation in the absence of α-crystallin (Das and Surewicz, 1995). Also, the addition of 1 % deoxycholate results in the formation of αA-crystallin tetrameric species. The addition of deoxycholate changes the environment surrounding the native aggregate by providing other hydrophobic surfaces with which the shielded hydrophobic regions of the α-crystallin subunits can interact. The lowest free energy form is one in which the hydrophobic portions are shielded from solvent. It is in this sense that the α-crystallin native aggregate is kinetically ‘ trapped ’. In regard to the long-term stability of population characteristics, the aggregation behavior of αcrystallin is quite similar to that of synthetic myosin filaments (Koretz, Coluccio and Kerrasso, 1982 ; Tumminia, Koretz and Landau, 1989). Like αcrystallin, synthetic myosin populations do not change over time, despite environmental conditions that would, during the assembly process, lead to characteristics very different from what is observed. Also like α-crystallin, the concentration of which myosin monomer alone is observed in solution is well below micromolar. Unlike α-crystallin, however, the basis for myosin’s aggregate stability, despite environmental changes, appears to be primarily steric in nature (Tumminia et al., 1989). This stability is due to a transition from a sheet-like structure to a rolled-up structure, in which most of the myosin molecule becomes inaccessible to solvent. It is possible that the underlying basis of the forces that stabilize the native α-crystallin aggregate is the subunit’s solubility characteristics in aqueous medium. At the present time, the only ways to dissociate the native particle are through urea or pH denaturation (Stevens and Augusteyn, 1993), or through the use of detergents (Radlick and Koretz, unpublished ; Horwitz, personal communication). The amphipathic nature of the subunit, as characterized by primary sequence analysis, detergent solubility, and other methods, is such that its lowest free energy form would coincide with partial or complete shielding of the hydrophobic regions of the subunit from the external medium. The relationship between protein concentration and solution turbidity for the denatured}renatured samples suggests that the smaller aggregates, particularly the monomer, are insoluble or only marginally soluble in buffer. With increasing aggregate size, increased shielding of

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hydrophobic regions may result. This shielding may yield increased solubilization in and interaction with water. Although the presumed hydrophobic regions of the α-crystallin subunit are shielded from solvent in the ‘ native ’ aggregate, these regions may still be accessible under the appropriate conditions. The functional testing of the chaperone-like activity of α-crystallin, before and after denaturation and renaturation, has confirmed that the aggregate is capable of providing γcrystallin with protection from heat denaturation and aggregation. If the mechanism by which α-crystallin protects γ-crystallin is analogous to that proposed for other molecular chaperones, the ‘ native ’ structure must be either sufficiently open or sufficiently flexible to provide hydrophobic surfaces which serve to stabilize secondary structure intermediates. In this respect, Boyle and Takemoto (1994) have shown that a macromolecule as large as an antibody is capable of binding to the N-terminal (hydrophobic) region of the α-crystallin subunit, despite its protected location within the aggregate, and that this region is centrally located within the aggregate. 5. Conclusions α-crystallin aggregates exhibit a number of characteristics that must be explained by any structural model. These characteristics include : heterogeneity in size and weight with an 800 kD average molecular mass ; equivalent microenvironments for every subunit ; lack of structural dependence on species-specific molar ratios between subunit isoforms ; and, flexibility in aggregate structure for access to hydrophobic surface(s) as part of the chaperone-like function. Of the models currently under proposal, the micellar model is more consistent with these properties than any of the others, but seems to fail because of the universally observed presence of the ‘ native ’ particle in solutions with concentrations well below that of the critical micelle concentration. The results presented here indicate that the ‘ native ’ aggregate may be, in fact, kinetically trapped into its configuration. Concentration-dependent aggregation studies, starting with denatured solutions of α-crystallin, demonstrate that small intermediates are formed below the CMC. These small intermediates coexist in equilibrium with the 800 kD aggregate, which begins to appear at the lower end of the CMC range. As the structure and properties of α-crystallin become better understood, it is likely that the micellar and other models will be supplanted by one that more accurately reflects the accumulated body of knowledge about it. For now, however, the micellar model provides both a convenient scaffold for the organization of the current state of information about αcrystallin and a testable hypothesis that can be used to lead to further characterization of α-crystallin’s biochemical and biophysical properties.

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Acknowledgements The support of NIH grant EY10011 for these and continuing studies on α-crystallin is gratefully acknowledged. The authors thank John C. Salerno for constructive discussions about detergents and micelles.

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