Biophysical characterization of alpha-crystallin aggregates: validation of the micelle hypothesis.

The size of alpha-crystallin aggregates, as well as the structural organization of each particle's subunits, is currently unknown, although a number of different laboratories have suggested both structures and average molecular weights (Thomson, J.A. and Augusteyn, R.C. (1984) Proc. Int. Soc. Eye Res. 3, 152). One hypothesis, compatible with literature reports and consistent with what is known of subunit primary structure and physiological function, is that alpha-crystallin exists in vivo as a naturally occurring protein micelle (Sen, A.C. and Chakrabarti, B. (1991) Biophysical J. 59, 108a.) To test this hypothesis, experiments were performed on this protein to determine its behavior under increased hydrostatic pressure and the effect of its concentration on aqueous surface tension. With increasing hydrostatic pressure, the turbidity of an alpha-crystallin solution increases exponentially to a plateau at about 6000-8000 psi; upon release of pressure, the samples slowly return to their original turbidity level. Other naturally aggregating proteins, such as skeletal muscle myosin, demonstrate a decrease in turbidity under the same conditions. The surface tension of alpha-crystallin in aqueous solution decreases to a plateau with increasing protein subunit concentration, with an inflection point over the range 0.18-0.25 mM; cholate and other amphiphiles exhibit similar behavior. In contrast, plots of surface tension over the equivalent concentration range for other protein aggregates in the same buffer more closely approximate the types of curve obtained with short chain aliphatic acids. These results indicate that alpha-crystallin behaves like the protein version of a micelle.     

Blue-fluorescent bovine alpha-crystallin

Blue-fluorescent alpha-crystallin has been isolated from bovine lenses by gel-filtration on Sephacryl S-200 Superfine. The blue fluorescence of this alpha-crystallin is characterized by fluorescence peaks at about 410 and 435 nm and two excitation peaks at about 350 and 370 nm. This finding suggests the existence of two different blue-fluorescences in bovine alpha-crystallin. Both low molecular weight alpha-crystallin and higher molecular weight alpha-crystallin exhibit similar blue fluorescence. With aging, in the nuclear region of bovine lenses, blue fluorescent low molecular weight alpha-crystallin shifts to non-covalently-linked higher molecular weight aggregates which are also blue-fluorescent.     

Lens proteasome shows enhanced rates of degradation of hydroxyl radical modified alpha-crystallin

Proteasome, a high molecular weight protease complex (HMP, approximately 600 kDa) was isolated from bovine eye lens epithelium tissue. In contrast with prior reports, lens proteasome degraded the major lens protein alpha-crystallin and S-carboxymethylated bovine serum albumin at 37 degrees C, mostly to trichloroacetic acid precipitable polypeptides. The proteasome, thus isolated, was labile at 55 degrees C. As indicated by the ability of p-chloromercuribenzoate and N-ethylmaleimide to block activity, a thiol group is required for activity. Alpha-crystallin was oxidized by exposure to 60Co-irradiation under an atmosphere of N2O (1-50 kilorads). This dose delivered 0.1-5.7 mol of hydroxyl radicals per mol of crystallin. Irradiation resulted in increased heterogeneity, aggregation, and fragmentation of the crystallin preparation. The proteolytic susceptibility of alpha-crystallin to the lens HMP was enhanced by the irradiation in a dose-dependent manner up to 20 kilorads (.OH concentration up to 2.3 mol per mol of alpha-crystallin). When 50 kilorads (5.7 mol .OH per mol of alpha-crystallin) was used, there was extensive aggregation and no enhancement in proteolysis over the unirradiated sample. The data indicate that the lens HMP can degrade mildly photooxidized lens proteins, but proteins which are extensively damaged are not degraded and may accumulate. This may be related to cataract formation.     

Studies of alpha- and betaL-crystallin complex formation in solution at 60 degrees C

Studies of molecular mechanisms of chaperone-like activity of alpha-crystallin became an active field of research over last years. However, fine interactions between alpha-crystallin and the damaged protein and their complex organization remain largely uncovered. Complexation between alpha- and betaL-crystallins was studied with thermal denaturation of betaL-crystallin at 60 degrees C using small-angle X-ray scattering (SAXS), light scattering, gel-permeation chromatography and electrophoresis. A mixed solution of alpha- and betaL-crystallins in concentrations about 10 mg/ml incubated at 60 degrees C was found to contain their soluble complexes with mean radius of gyration approximately 14 nm, mean molecular weight approximately 4000 kDA and maximal size approximately 40 nm. In pure betaL-crystallin solution, complexes were not observed at 60 degrees C. In SAXS studies, transitions in the alpha-crystallin quaternary structure at 60 degrees C were shown to occur and result in a double increase of the molecular weight. It suggests that during the temperature-induced denaturation of betaL-crystallin it binds with modified alpha-crystallin or, alternatively, alpha-betaL-crystallin complexation and alpha-crystallin modifications are concurrent. Estimates of the alpha-betaL-crystallin dimensions and relative contents of alpha- and betaL-crystallins in the complex suggest that several alpha-crystallin molecules are involved in complex formation.     

Native quaternary structure of bovine alpha-crystallin

Alpha-crystallin is the most important soluble protein in the eye lens. It is responsible for creating a high refractive index and is known to be a small heat-shock protein. We have used static and dynamic light scattering to study its quaternary structure as a function of isolation conditions, temperature, time, and concentration. We have used tryptophan fluorescence to study the temperature dependence of the tertiary structure and its reversibility. Gel filtration, analytical ultracentrifugation, polyacrylamide gel electrophoretic analysis, and absorption measurements were used to study the chaperone-like activity of alpha-crystallin in the presence of destabilized lysozyme. We have demonstrated that the molecular mass of the in vivo alpha-crystallin oligomer is about 700 kDa (alpha(native)) while the 550 kDa molecule (alpha(37 degrees C),diluted), which is often found in vitro, is a product of prolonged storage at 37 degrees C of low concentrated alpha-crystallin solutions. We have proven that the molecular mass of the alpha-crystallin oligomer is concentration dependent at 37 degrees C. We have found strong indications that, during chaperoning, the alpha-crystallin oligomer undergoes a drastic rearrangement of its peptides during the process of complex formation with destabilized lysozyme. We propose the hypothesis that all these processes are governed by the phenomenon of subunit exchange, which is well-known to be strongly temperature-dependent.     

A small-angle X-ray solution scattering study of bovine alpha-crystallin.

The native high molecular mass form of alpha-crystallin, the most important soluble protein in the eye lens, and its low molecular mass form obtained at 37 degrees C in dilute solutions were investigated by synchrotron radiation small-angle X-ray scattering. The alpha-crystallin solutions are polydisperse and good fits to the experimental data can be obtained using distributions of spheres with radii varying between about 5 and 10 nm. In spite of the polydispersity, two different ab initio methods were used to retrieve low resolution shapes from the scattering data. These shapes correspond to the z-average structure of the oligomers. In the absence of any symmetry constraints, the scattering curves of the two forms of alpha-crystallin yield bean-like shapes. The shape corresponding to the low molecular mass form has about 20% less mass at the periphery. Imposing tetrahedral symmetry on the average structures worsens the fit to the experimental data. We emphasized the apparent contradiction between hydrodynamic and molecular properties of alpha-crystallin. An explanation was put forward based on the presence of solvent-exposed flexible C-terminal extensions. We present two bead models ('hollow globule with tentacles' and 'bean with tentacles') based on NMR and cryo-electron microscopy studies and discuss how well they correspond with our data from X-ray scattering, light scattering and analytical ultracentrifugation.     

Complete protection by alpha-crystallin of lens sorbitol dehydrogenase undergoing thermal stress

Sorbitol dehydrogenase (l-iditol:NAD(+) 2-oxidoreductase, E.C. 1.1.1. 14) (SDH) was significantly protected from thermally induced inactivation and aggregation by bovine lens alpha-crystallin. An alpha-crystallin/SDH ratio as low as 1:2 in weight was sufficient to preserve the transparency of the enzyme solution kept for at least 2 h at 55 degrees C. Moreover, an alpha-crystallin/SDH ratio of 5:1 (w/w) was sufficient to preserve the enzyme activity fully at 55 degrees C for at least 40 min. The protection by alpha-crystallin of SDH activity was essentially unaffected by high ionic strength (i.e. 0.5 m NaCl). On the other hand, the transparency of the protein solution was lost at a high salt concentration because of the precipitation of the alpha-crystallin/SDH adduct. Magnesium and calcium ions present at millimolar concentrations antagonized the protective action exerted by alpha-crystallin against the thermally induced inactivation and aggregation of SDH. The lack of protection of alpha-crystallin against the inactivation of SDH induced at 55 degrees C by thiol blocking agents or EDTA together with the additive effect of NADH in stabilizing the enzyme in the presence of alpha-crystallin suggest that functional groups involved in catalysis are freely accessible in SDH while interacting with alpha-crystallin. Two different adducts between alpha-crystallin and SDH were isolated by gel filtration chromatography. One adduct was characterized by a high M(r) of approximately 800,000 and carried exclusively inactive SDH. A second adduct, carrying active SDH, had a size consistent with an interaction of the enzyme with monomers or low M(r) aggregates of alpha-crystallin. Even though it had a reduced efficiency with respect to alpha-crystallin, bovine serum albumin was shown to mimic the chaperone-like activity of alpha-crystallin in protecting SDH from thermal denaturation. These findings suggest that the multimeric structural organization of alpha-crystallin may not be a necessary requirement for the stabilization of the enzyme activity.     

Glycation by ascorbic acid oxidation products leads to the aggregation of lens proteins

Previous studies from this laboratory have shown that there are striking similarities between the yellow chromophores, fluorophores and modified amino acids released by proteolytic digestion from calf lens proteins ascorbylated in vitro and their counterparts isolated from aged and cataractous lens proteins. The studies reported in this communication were conducted to further investigate whether ascorbic acid-mediated modification of lens proteins could lead to the formation of lens protein aggregates capable of scattering visible light, similar to the high molecular aggregates found in aged human lenses. Ascorbic acid, but not glucose, fructose, ribose or erythrulose, caused the aggregation of calf lens proteins to proteins ranging from 2.2 x 10(6) up to 3.0 x 10(8 )Da. This compared to proteins ranging from 1.8 x 10(6) up to 3.6 x 10(8 )Da for the water-soluble (WS) proteins isolated from aged human lenses. This aggregation was likely due to the glycation of lens crystallins because [U-(14)C] ascorbate was incorporated into the aggregate fraction and because NaCNBH(3), which reduces the initial Schiff base, prevented any protein aggregation. Reactions of ascorbate with purified crystallin fractions showed little or no aggregation of alpha-crystallin, significant aggregation of beta(H)-crystallin, but rapid precipitation of purified beta(L)- and gamma-crystallin. The aggregation of lens proteins can be prevented by the binding of damaged crystallins to alpha-crystallin due to its chaperone activity. Depending upon the ratios between the components of the incubation mixtures, alpha-crystallin prevented the precipitation of the purified beta(L)- and gamma-crystallin fractions during ascorbylation. The addition of at least 20% of alpha-crystallin by weight into glycation mixtures with beta(L)-, or gamma-crystallins completely inhibited protein precipitation, and increased the amount of the high molecular weight aggregates in solution. Static and dynamic light scattering measurements of the supernatants from the ascorbic acid-modified mixtures of alpha- and beta(L)-, or gamma-crystallins showed similar molar masses (up to 10(8 )Da) and hydrodynamic diameter (up to 80( )nm). These data support the hypothesis, that if the lens reducing environment is compromised, the ascorbylation of lens crystallins can significantly change the short range interactions between different classes of crystallins leading to protein aggregation, light scattering and eventually to senile cataract formation.     

The selective inhibition of serpin aggregation by the molecular chaperone, alpha-crystallin, indicates a nucleation-dependent specificity

Small heat shock proteins (sHsps) are a ubiquitous family of molecular chaperones that prevent the misfolding and aggregation of proteins. However, specific details about their substrate specificity and mechanism of chaperone action are lacking. alpha1-Antichymotrypsin (ACT) and alpha1-antitrypsin (alpha1-AT) are two closely related members of the serpin superfamily that aggregate through nucleation-dependent and nucleation-independent pathways, respectively. The sHsp alpha-crystallin was unable to prevent the nucleation-independent aggregation of alpha1-AT, whereas alpha-crystallin inhibited ACT aggregation in a dose-dependent manner. This selective inhibition of ACT aggregation coincided with the formation of a stable high molecular weight alpha-crystallin-ACT complex with a stoichiometry of 1 on a molar subunit basis. The kinetics of this interaction occur at the same rate as the loss of ACT monomer, suggesting that the monomeric species is bound by the chaperone. 4,4'-Dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (Bis-ANS) binding and far-UV circular dichroism data suggest that alpha-crystallin interacts specifically with a non-native conformation of ACT. The finding that alpha-crystallin does not interact with alpha1-AT under these conditions suggests that alpha-crystallin displays a specificity for proteins that aggregate through a nucleation-dependent pathway, implying that the dynamic nature of both the chaperone and its substrate protein is a crucial factor in the chaperone action of alpha-crystallin and other sHsps.     

Subunit exchange demonstrates a differential chaperone activity of calf alpha-crystallin toward beta LOW- and individual gamma-crystallins

The chaperone activity of native alpha-crystallins toward beta(LOW)- and various gamma-crystallins at the onset of their denaturation, 60 and 66 degrees 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 degrees C. Further increase is observed at 66 degrees 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 degrees C, the rate of exchange was increased depending upon the temperature of incubation, 60 or 66 degrees C. Association with beta(LOW)- (60 degrees C) or various gamma-crystallins (66 degrees C) resulted at 37 degrees 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 h gamma 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.     

Self-similarity properties of alpha-crystallin supramolecular aggregates.

The supramolecular aggregation of alpha-crystallin, the major protein of the eye lens, was investigated by means of static and dynamic light scattering. The aggregation was induced by generating heat-modified alpha-crystallin forms and by stabilizing the clusters with calcium ions. The kinetic pattern of the aggregation and the structural features of the clusters can be described according to the reaction limited cluster-cluster aggregation theory previously adopted for the study of colloidal particles aggregation systems. Accordingly, the average mass and the hydrodynamic radius of alpha-crystallin supramolecular aggregates grow exponentially in time. The structure factor of the clusters is typical of fractal aggregates. A fractal dimension df approximately 2.15 was determined, indicating a low probability of sticking together of the primitive aggregating particles. As a consequence, the slow-forming clusters assemble a rather compact structure. The basic units forming the fractal aggregates were found to have a radius about twice (approximately 17 nm) that of the native protein and 5.3 times its size, which is consistent with an intermediate molecular assembly corresponding to the already known high molecular weight forms of alpha-crystallin.     

alpha-Crystallin chaperone-like activity and membrane binding in age-related cataracts.

alpha-Crystallin, the major protein component of the vertebrate lens, is thought to play a critical role in the maintenance of transparency through its ability to inhibit stress-induced protein aggregation. However, during aging and cataract formation the amount of membrane-bound alpha-crystallin increases significantly while high molecular weight complexes (HMWCs) comprised of alpha-crystallin and other lens crystallins accumulate. These and other recent data suggest a possible link between cataract formation, the formation of high molecular weight alpha-crystallin aggregates, and the progressive increase in membrane association of alpha-crystallin. To better understand these processes, we characterized the chaperone-like activity (CLA) and subunit exchange of membrane bound alpha-crystallin. In addition, we measured the membrane binding properties of in vitro constituted HMWCs to understand the mechanism by which increased alpha-crystallin is bound to the membrane of old and cataractous lens cells in vivo. Membrane-associated alpha-crystallin complexes have measurably reduced CLA compared to complexes in solution; however, membrane binding does not alter the time required for alpha-crystallin complexes to reach subunit exchange equilibrium. In addition, HMWCs prepared in vitro have a profoundly increased membrane binding capacity as compared to native alpha-crystallin. These results are consistent with a model in which increased membrane binding of alpha-crystallin is an integral step in the pathogenesis of many forms of cataracts.     

Inhibition of alpha-crystallin aggregation by gamma-crystallin

The transparency of the mammalian lens is primarily maintained by short range order among the major proteins of the lens fiber cells, the crystallins. Although these proteins are highly conserved at the amino acid sequence level, it has proven difficult to establish that they possess other than structural functions. We find that when non-lens proteins are added to concentrated solutions of alpha-crystallin, aggregation is induced, presumably through excluded volume effects. In contrast, the monomeric gamma-crystallins and the low molecular weight form of beta-crystallin (beta L) cause a decrease in the size of alpha-crystallin. When the naturally aggregated form of alpha-crystallin is examined, gamma- and beta L-crystallin, as well as a reducing agent, also cause partial dissociation as detected by dynamic light scattering and size exclusion chromatography, while no effect is seen with non-crystallin proteins. Furthermore, the chemical cross-linking of alpha-crystallin is inhibited by gamma- and beta L-crystallin but not by other proteins. The ability of gamma-crystallin to inhibit the association of alpha-crystallin is primarily localized to the gamma-II form which contains a high degree of exposed thiols. Only small amounts of gamma- and beta L-crystallin, however, can be cross-linked to alpha-crystallin in mixtures of the three proteins even at very high protein concentrations. These results suggest that one possible role for the lower molecular weight crystallins may be to minimize through a reductive effect the intrinsic tendency of alpha-crystallin to aggregate, an association reaction implicated in the loss of lens transparency.     

Structural changes in alpha-crystallin and whole eye lens during heating, observed by low-angle X-ray diffraction

Whole eye lens and alpha-crystallin gels and solutions were investigated using X-ray scattering techniques at temperatures ranging from 20 degrees C to 70 degrees C. In whole lens isolated in phosphate-buffered saline, the spacing of the dominant X-ray reflection seen with low-angle scattering was constant from 20 degrees C to 45 degrees C but increased at 50 degrees C from 15.2 nm to 16.5 nm. At room temperature, the small-angle X-ray diffraction pattern of the intact lens was very similar to the pattern of alpha-crystallin gels at near-physiological concentration (approximately 300 mg/ml), so it is reasonable to assume that the alpha-crystallin pattern dominates the pattern of the intact lens. Our results therefore indicate that in whole lens alpha-crystallin is capable of maintaining its structural properties over a wide range of temperature. This property would be useful in providing protection for other lens proteins super-aggregating. In the alpha-crystallin gels, a moderate increase in both the spacing and intensity of the reflection was observed from 20 degrees C to 45 degrees C, followed by an accelerated increase from 45 degrees C to 70 degrees C. Upon cooling, this effect was found to be irreversible over 11 hours. Qualitatively similar results were observed for alpha-crystallin solutions at a variety of lower concentrations.     

Second virial coefficient of alpha-crystallin

Light scattering studies were performed on bovine alpha-crystallin measuring the scattering intensities as a function of scattering angle, concentration, and temperature. The data yielded the molecular weight, radius of gyration, and second virial coefficient of alpha-crystallin at different temperatures. The second virial coefficient increased with increasing temperature. Both the enthalpy and entropy of solution of alpha-crystallin are positive. The Flory theta temperature was found to be 271 K.     

Study of the chaperoning mechanism of bovine lens alpha-crystallin, a member of the alpha-small heat shock superfamily

We have studied the interaction between lysozyme, destabilized by reducing its -S-S- bonds, and bovine eye lens alpha-crystallin, a member of the alpha-small heat shock protein superfamily. We have used gel filtration, photon correlation spectroscopy, and analytical ultracentrifugation to study the binding of lysozyme by alpha-crystallin at 25 degrees C and 37 degrees C. We can conclude that alpha-crystallin chaperones the destabilized protein in a two-step process. First the destabilized proteins are bound by the alpha-crystallin so that nonspecific aggregation of the destabilized protein is prevented. This complex is unstable, and a reorganization and inter-particle exchange of the peptides result in stable and soluble large particles. alpha-Crystallin does not require activation by temperature for the first step of its chaperone activity as it prevents the formation of nonspecific aggregates at 25 degrees C as well as at 37 degrees C. The reorganization of the peptides, however, gives rise to smaller particles at 37 degrees C than at 25 degrees C. Indirect evidence shows that the association of several alpha-crystallin/substrate protein complexes leads to the formation of very large particles. These are responsible for the increase of the light scattering.     

Interaction between the constituent A and B lens alpha-crystallin subunits leading to formation of alpha-neoprotein molecules

A and B constituent subunits associated in lens alpha-crystallin were found to interact with added B chains forming alpha-neoprotein molecules with lower A to B chain ratios than 2 A to 1 B in alpha-crystallin. Addition of 1% excess of B chains to the one in alpha-crystallin, which resulted in a ratio of 1.98 A to 1 B in the mixture, caused a change of quaternary structure in 30% of alpha-crystallin molecules within 18 h. At a ratio of 1.86 A to 1 B, all alpha-crystallin molecules were affected at this time. A maximum number of 495 B chains was found to form an association with 1 A chain, initially bound in alpha-crystallin. Such a high number may indicate that the reaction involves monomeric A chains binding aggregated macromolecules of B chains. It is in such form that B chains occur as macromolecules with an average molecular weight of 0.7 X 10(6) in aqueous solution. The alpha-neoprotein molecules selected for studies in this report had A to B chain ratios of 1.75:1, 1:1, and 0.2:1. Each behaved in immunodiffusion tests like single molecular entities. Antigenic determinants located on A as well as on B chains associated with each other in alpha-crystallin were found to be identical with determinants on the chains associated in the above alpha-neoprotein molecules. Determinants dependent on the quaternary structure of alpha-neoprotein and of alpha-crystallin molecules were completely different. Some of the quaternary determinants of various alpha-neoproteins were type specific and did not occur in molecules with different A to B chain ratios. Other quaternary determinants occurred in all alpha-neoproteins. An excess of A chains did not revert alpha-neoproteins to alpha-crystallin. However, alpha-neoprotein molecules did interact with added B chains forming neomolecules with lower A to B chain ratios.     

The alphaA-crystallin R116C mutant has a higher affinity for forming heteroaggregates with alphaB-crystallin.

An autosomal dominant congenital cataract in humans is associated with mutation of Arg-116 to Cys in alphaA-crystallin (alphaA-R116C). The chaperone activity and biophysical properties of reconstituted alpha-crystallin from different proportions of wild-type alphaB-crystallin (alphaB-wt) and alphaA-R116C-crystallin were studied by gel permeation chromatography, SDS-polyacrylamide gel electrophoresis, and fluorescence and circular dichroism spectroscopy and compared with those of reconstituted alpha-crystallin from alphaB-wt and wild-type alphaA-crystallin (alphaA-wt). The reconstituted alpha-crystallin containing alphaA-R116C and alphaB-wt had a higher molecular mass, a higher thermal sensitivity to exposition of Trp side chains, fewer available hydrophobic surfaces, and lower chaperone activity than the alpha-crystallin containing alphaA-wt and alphaB-wt. The secondary structure exhibited very small changes, whereas the tertiary structure was distinctly different for alpha-crystallin formed from alphaA-R116C and alphaB-wt. Most importantly, subunit exchange studies by fluorescence resonance energy transfer showed that alphaA-R116C forms heteroaggregates faster than alphaA-wt with alphaB-wt, and the reconstituted alpha-crystallins were true heteroaggregates of two interacting subunits. These findings suggest that the molecular basis for the congenital cataract with the alphaA-R116C mutation is the formation of highly oligomerized heteroaggregates of alpha-crystallin with modified structure. However, contrary to the earlier conclusions based on the studies of homoaggregates, the loss in chaperone activity of the heteroaggregates having alphaA-R116C does not appear to be large enough to become the main factor in initiating cataract development in the affected individuals.     

Significance of alpha-crystallin heteropolymer with a 3:1 alphaA/alphaB ratio: chaperone-like activity, structure and hydrophobicity

The small heat-shock protein alpha-crystallin isolated from the eye lens exists as a large (700 kDa) heteropolymer composed of two subunits, alphaA and alphaB, of 20 kDa each. Although trace amounts of alphaA-crystallin are found in other tissues, non-lenticular distribution of alpha-crystallin is dominated by the alphaB homopolymer. In most vertebrate lens, the molar ratio of alphaA to alphaB is generally 3:1. However, the importance of this ratio in the eye lens is not known. In the present study, we have investigated the physiological significance of the 3:1 ratio by determining the secondary/tertiary structure, hydrophobicity and chaperone-like activity of alphaA- and alphaB-homopolymers and heteropolymers with different ratios of alphaA to alphaB subunits. Although, under physiologically relevant conditions, the alphaB-homopolymer (37-40 degrees C) has shown relatively higher activity, the alphaA-homopolymer or the heteropolymer with a higher alphaA proportion (3:1 ratio) has shown greater chaperone-like activity at elevated temperatures (>50 degrees C) and also upon structural perturbation. Furthermore, higher chaperone activity at elevated temperatures as well as upon structural perturbation is mainly mediated through increased hydrophobicity of alphaA. Although homopolymers and heteropolymers of alpha-crystallin did not differ in their secondary structure, changes in tertiary structure due to structural perturbations upon pre-heating are mediated predominantly by alphaA. Interestingly, the heteropolymer with higher alphaA proportion (3:1) or the alphaA-homopolymer seems to be better chaperones in protecting lens beta- and gamma-crystallins at both normal and elevated temperatures. Thus lens might have favoured a combination of these qualities to achieve optimal protection under both native and stress (perturbed) conditions for which the heteropolymer with alphaA to alphaB in the 3:1 ratio appears to be better suited.     

Further studies on the sub-units of α-crystallin

1. A new procedure is described for the purification of alpha-crystallin, including: preparative zone electrophoresis, density-gradient centrifugation and gel filtration. The total amino acid composition of highly purified samples prepared according to this procedure has been determined. 2. Evidence is presented for the presence of intermediates in the urea-induced splitting of alpha-crystallin into sub-units. A possible mechanism for this splitting is proposed. 3. The recombination of sub-units has been studied by polyacrylamide-gel electrophoresis and ultracentrifugal analysis. As judged from these criteria, only a partial recovery of starting material was obtained. 4. The origin of the minor bands in the electrophoretic pattern of alpha-crystallin on 7m-urea-polyacrylamide gel has been investigated. No evidence was found that their presence is due to carbamoylation or sulphide-disulphide interchange. They probably arise from isomerization. 5. The mean molecular weight of the sub-units was calculated to be 24000 (Archibald's method). Determination of the sedimentation-diffusion equilibrium revealed a value of 21000 at the meniscus. Assuming that all sub-units contain one cysteine residue/molecule, 23000 can be derived for the mean molecular weight.