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.     

Calf lens alpha-crystallin quaternary structure. A three-layer tetrahedral model

Calf lens alpha-crystallins are polydisperse globular particles made of a large number of two types of subunits, A and B, both of molecular weight congruent to 20,000. alpha-Crystallin populations consisting on average of 40 subunits or more were subjected to various changes in pH, ionic strength, temperature and urea concentration. Modifications in quaternary structure induced by variation of these physicochemical parameters were followed by means of X-ray and quasi-elastic light-scattering and quantified in terms of weight average molecular weight (M), radius of gyration (Rg) and hydrodynamic radius (Rh). High-pressure liquid chromatography was used as a control of polydispersity. Increasing the pH, decreasing the ionic strength and incubating at temperatures from 20 degrees C to 45 degrees C all resulted in the formation of particles of decreasing M, Rg and Rh values. These effects are cumulative. All monomodal alpha-crystallin populations encountered in this study, which covers a wide range of sizes and molecular weights, may be accounted for by a three-layer model with partial filling up of the layers. Applying basic principles of symmetry and postulating specific contacts between protein subunits to construct this three-layer model leads to tetrahedral symmetry, with 12, 24 and 24 sites in the first, second and third layers, respectively. Variations in probabilities of site occupancy account for both the observed quaternary structure modifications and the intrinsic polydispersity of alpha-crystallins     

Folding-unfolding and aggregation-dissociation of bovine alpha-crystallin subunits; evidence for unfolding intermediates of the alpha A subunits

The aggregation and dissociation behavior of bovine alpha-crystallin as well as the folding and unfolding of its subunits were investigated by equilibrium studies using tryptophan fluorescence measurements and two isoelectric focusing techniques, viz. isoelectric focusing across a urea gradient and isoelectric focusing in two dimensions with different concentrations of urea. It was found that the alpha B chains lose their ability to aggregate and start unfolding at a lower concentration of urea than the alpha A chains. Equilibrium intermediates were found upon unfolding or refolding of alpha A subunits, which can be explained by a two-domain organization of these molecules.     

A novel quaternary structure of the dimeric alpha-crystallin domain with chaperone-like activity

alphaB-crystallin, a member of the small heat-shock protein family and a major eye lens protein, is a high molecular mass assembly and can act as a molecular chaperone. We report a synchrotron radiation x-ray solution scattering study of a truncation mutant from the human alphaB-crystallin (alphaB57-157), a dimeric protein that comprises the alpha-crystallin domain of the alphaB-crystallin and retains a significant chaperone-like activity. According to the sequence analysis (more than 23% identity), the monomeric fold of the alpha-crystallin domain should be close to that of the small heat-shock protein from Methanococcus jannaschii (MjHSP16.5). The theoretical scattering pattern computed from the crystallographic model of the dimeric MjHSP16.5 deviates significantly from the experimental scattering by the alpha-crystallin domain, pointing to different quaternary structures of the two proteins. A rigid body modeling against the solution scattering data yields a model of the alpha-crystallin domain revealing a new dimerization interface. The latter consists of a strand-turn-strand motif contributed by each of the monomers, which form a four-stranded, antiparallel, intersubunit composite beta-sheet. This model agrees with the recent spin labeling results and suggests that the alphaB-crystallin is composed by flexible building units with an extended surface area. This flexibility may be important for biological activity and for the formation of alphaB-crystallin complexes of variable sizes and compositions.     

Specific dissociation of alpha B subunits from alpha-crystallin

Exposure of bovine alpha-crystallin to 0.1 M glycine at pH 7 decreases the average molar mass of the protein from 700 to 420 kDa. When the pH is lowered to 2.5, in the same buffer, the alpha B chains specifically dissociate from the aggregates, leaving a particle of 290 kDa containing only alpha A chains. The decrease in the molar mass corresponds to the mass of the alpha B chains in the original aggregate. The pH-dependent dissociation is fully reversible. Similar changes were observed with rat and kangaroo alpha-crystallins but the dogfish protein was not affected. Sedimentation velocity analyses and fluorescence spectroscopy yielded a pK, for the dissociation, of 3.7 for alpha-crystallin and 4.0 for a homopolymer constructed from purified alpha B2 polypeptides. An alpha A2 homopolymer was virtually unaffected by the lowering of pH. The products from the dissociation were isolated and their properties studied by sedimentation analysis and acrylamide quenching of tryptophan fluorescence. The alpha B chains were found to be completely denatured, whereas the structure of the alpha A chains, in the 290 kDa, particle, were only slightly altered. Comparisons of the sequences of the various proteins examined suggested that decreased ionization of aspartic acid 127 in the alpha B chain was responsible for the specific dissociation of this polypeptide.     

A structural study of bovine lens alpha-crystallin and its subunits by absorption and linear dichroism spectroscopy

The structure of the bovine alpha-crystallin aggregate and its reaggregated isolated subunits has been studied by measurement of their absorption and linear dichroism spectra over the range 250-350 nm. Also, changes in structure with respect to time have been monitored in this way. From the absorption spectra it appears that the aromatic residues in subunit aggregates are in the same chemical environment as those in native protein. The light scattering due to the size of the protein molecules increases when the proteins are kept in solution, this effect being much stronger for the subunits. The linear dichroism spectra point to strong structural ordering in alpha-crystallin, the absorption transition dipoles of the aromatic residues being preferentially aligned along the long axis of the molecules. Moreover, a considerable deviation from a spherical or tetrahedrally symmetric structure of alpha-crystallin is inferred. The subunit aggregates show less ordering and might be more spherical. When kept in solution, their structural order seems to be decreased. The linear dichroism spectra show absorption at 325 nm, which is not detectable in the normal absorption spectra.     

Formation of native structure by intermolecular thiol-disulfide exchange reactions without oxidants in the folding of bovine pancreatic ribonuclease A

It has been shown previously that the oxidative folding of bovine pancreatic ribonuclease A proceeds through parallel pathways with two major native-like three-disulfide (3S) intermediates. We show here that, under some conditions, the native disulfide bonds can also be regenerated through disproportionation reactions; in other words, the protein can serve as its own redox reagent. The results also show that disulfide species of the unstructured 3S ensemble have a strong propensity to participate in intermolecular interactions. These interactions are favored at high protein concentration, temperature and pH, and lead to formation of the native structure during disulfide reshuffling in the rate-determining step.     

Effect of hydrostatic pressure on quaternary structure on the chaperone function of alpha-crystallin

High hydrostatic pressure-induced changes in bovine lens alpha-crystallin oligomers size and chaperone-like function were studied by a static light scattering. Under pressure 1.5 kbar, alpha-crystallin oligomers size is almost unaffected. Increase of the size was observed during several hours of incubation at 3 kbar. Such high-pressure effect on association has been previously revealed for detergent micelles, whereas the "typical" protein oligomers are known to dissociate under high pressure. Our results about pressure influence on alpha-crystallin association supports the previously proposed "protein micelle" model of the protein quaternary structure. Chaperone-like activity of alpha-crystallin is shown to increase after incubation at 3 kbar. After the end of the incubation this activity is slowly decreasing during several hours.     

The quaternary structure of bovine brain kinesin.

In the present work we have studied the subunit composition of kinesin, the microtubule-activated, mechanochemical ATPase, isolated from bovine brain. Polypeptides with mol. wts of 120 and 62 kd are the major components of the kinesin preparation. These polypeptides could not be separated by electrophoresis under nondenaturing conditions or by FPLC on a MonoQ column, and are therefore assumed to form a tight complex. As shown by immunoblotting with polyclonal and monoclonal antibodies to the 120-kd polypeptide and by one-dimensional peptide mapping, the 62-kd polypeptide does not appear to be a proteolytic product of the 120-kd component. Densitometric scanning of polyacrylamide-SDS gels shows that these polypeptides are present in a complex in a 1:1 molar ratio. The mol. wt of native kinesin was studied by sedimentation equilibrium and was found to be 386 +/- 14 kd. A comparison of the mol. wts of individual polypeptides with the mol. wt of the intact molecule indicates that the native molecule contains two 120-kd subunits and two 62-kd subunits.     

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.     

The quaternary structure of bovine heart mitochondrial creatine kinase

Crosslinking of subunits of the high molecular weight oligomer of bovine heart mitochondrial creatine kinase (CKm) by dimethyl suberimidate and subsequent electrophoresis in the presence of sodium dodecyl sulfate gives eight protein bands. An increase in the time course of the enzyme crosslinking reaction results in the protein accumulation in the high molecular weight bands. Evidence has been obtained suggesting that crosslinking involves only the intraoligomeric contact areas. It is concluded that bovine heart CKm is an octamer. Crosslinking of intersubunit contacts in the octameric form of the enzyme by various diimidates has been carried out. The data obtained suggest that within the octamer the CKm subunits have a quasispherical rather than planar arrangement. This finding is supported by electron microscopy data.     

Antisera to synthetic peptides as probes of structural changes during aging of alpha-crystallin from the bovine lens

Polyclonal antisera have been made to synthetic peptides of 11-15 residues that correspond to nine different regions of the alpha A crystallins. These antisera have been used in a radioimmunoassay to quantitatively probe for structural and/or covalent changes of alpha-crystallins in the nucleus versus cortex of the adult bovine lens. Antisera specific for the C-terminal and N-terminal regions of the alpha-crystallins bind more to alpha-crystallins from cortex. Antisera to three out of the seven internal sequences (residues 75-89, 87-101 and 135-149) bind better to alpha-crystallins from the bovine lens nucleus, suggesting a greater accessibility of these sequences to antisera binding. Together, these studies demonstrate that antisera against synthetic peptide sequences of alpha A crystallins are very specific probes that can detect structural and/or covalent changes in specified regions of the alpha-crystallins during the process of aging in the bovine lens.     

Prediction of quaternary structure from primary structure

MOTIVATION: The 'sequence implies conformation' principle has been the motivation for the construction of numerous systems of secondary and tertiary structure prediction. Computational experiments have shown that this principle can now be extended to quaternary structure prediction. This work appears to be the first effort to predict quaternary structure properties from sequence. RESULTS: The software developed to conduct these experiments was the Quaternary Structure Explorer (QSE). Successful rule-based classifiers have been found that can discriminate between the primary sequences of homodimers and non-homodimers.     

Molecular mass of macromolecules and subunits and the quaternary structure of hemoglobin from the microcrustacean Daphnia magna

The molecular masses of macromolecules and subunits of the extracellular hemoglobin from the fresh-water crustacean Daphnia magna were determined by analytical ultracentrifugation, multiangle laser light scattering and electrospray ionization mass spectrometry. The hemoglobins from hypoxia-incubated, hemoglobin-rich and normoxia-incubated, hemoglobin-poor Daphnia magna were analyzed separately. The sedimentation coefficient of the macromolecule was 17.4 +/- 0.1 S, and its molecular mass was 583 kDa (hemoglobin-rich animals) determined by AUC and 590.4 +/- 11.1 kDa (hemoglobin-rich animals) and 597.5 +/- 49 kDa (hemoglobin-poor animals), respectively, determined by multiangle laser light scattering. Measurements of the hemoglobin subunit mass of hemoglobin-rich animals by electrospray ionization mass spectrometry revealed a significant peak at 36.482 +/- 0.0015 kDa, i.e. 37.715 kDa including two heme groups. The hemoglobin subunits are modified by O-linked glycosylation in the pre-A segments of domains 1. No evidence for phosphorylation of hemoglobin subunits was found. The subunit migration behavior during SDS/PAGE was shown to be influenced by the buffer system used (Tris versus phosphate). The subunit mass heterogeneity found using Tris buffering can be explained by glycosylation of hemoglobin subunits. Based on molecular mass information, Daphnia magna hemoglobin is demonstrated to consist of 16 subunits. The quaternary structure of the Daphnia magna hemoglobin macromolecule was assessed by three-dimensional reconstructions via single-particle analysis based on negatively stained electron microscopic specimens. It turned out to be much more complex than hitherto proposed: it displays D4 symmetry with a diameter of approximately 12 nm and a height of about 8 nm.     

epsilon-crystallin from duck eye lens comparison of its quaternary structure and stability with other lactate dehydrogenases and complex formation with alpha-crystallin.

Taxon-specific epsilon-crystallin (epsilonC) from duck eye lens is identical to duck heart muscle lactate dehydrogenase. It forms a dimer of dimers with a dissociation constant of 2.2 x 10-7 M, far beyond the value observed for other vertebrate lactate dehydrogenases. Comparing the characteristics of wild-type epsilon-crystallin with those of three mutants, G115N, G119F and 115N/119F, representing the only significant peripheral sequence variations between duck epsilonC and chicken or pig heart muscle lactate dehydrogenase, no significant conformational differences are detectable. Regarding the catalytic properties, the Michaelis constant of the double mutant 115N/119F for pyruvate is found to be decreased; for wild-type enzyme, the effect is overcompensated by the high expression level of epsilonC in the eye lens. As taken from spectral analysis of the guanidine-induced and temperature-induced denaturation transitions, epsilonC in its dimeric state is relatively unstable, whereas the native tetramer exhibits the high intrinsic stability characteristic of common vertebrate heart and muscle lactate dehydrogenases. The denaturation mechanism of epsilonC is complex and only partially reversible. In the case of thermal unfolding, the predominant side reaction competing with the reconstitution of the native state is the kinetic partitioning between proper folding and aggregation. alpha-Crystallin, the major molecular chaperone in the eye lens, inhibits the aggregation of epsilonC by trapping the misfolded protein.