Quaternary Structure Of Alpha-crystallin Research Articles

Heat-induced structural transitions of alpha-crystallin studied by small-angle neutron scattering

Alpha-crystallin from the bovine eye lens was studied by small-angle neutron scattering (SANS) in 90% D2O buffer solution at 20, 50, 60, 65, 75, 85 and 95°C. The temperature points for this study were specified on the basis of differential scanning calorimetric analysis of alpha-crystallin solutions which has shown two endothermic transitions with midpoints at 64.5 and 86°C. The SANS study revealed no significant alpha-crystallin quaternary structure alterations at 50°C as compared with 20°C. At 60-65°C the SANS data confirmed substantial alpha-crystallin quaternary structure rearrangements which resulted in the formation of alpha-crystallin oligomers with a similar shape but approximately twofold increased molecular weight as compared to the native state at 20°C. At higher temperatures (75, 85 and 95°C) the SANS patterns were very similar and were consistent with the scattering by rod-like particles with a cross-section radius of gyration ∼55 This transformation of alpha-crystallin to the rod-like particles was evidently irreversible as these particles remained in solution after cooling to 20°C. Ab initio shape models of the native and high-temperature alpha-crystallin were retrieved with DAMMIN and DAMAVER software. Schematic model of alpha-crystallin heat-induced quaternary structure transitions was considered.

Evidence for specific subunit distribution and interactions in the quaternary structure of α‐crystallin

The quaternary structure of alpha-crystallin is dynamic, a property which has thwarted crystallographic efforts towards structural characterization. In this study, we have used collision-induced dissociation mass spectrometry to examine the architecture of the polydisperse assemblies of alpha-crystallin. For total alpha-crystallin isolated directly from fetal calf lens using size-based chromatography, the alphaB-crystallin subunit was found to be preferentially dissociated from the oligomers, despite being significantly less abundant overall than the alphaA-crystallin subunits. Furthermore, upon mixing molar equivalents of purified alphaA- and alphaB-crystallin, the levels of their dissociation were found to decrease and increase, respectively, with time. Interestingly though, dissociation of subunits from the alphaA- and alphaB-crystallin homo-oligomers was comparable, indicating that strength of the alphaA:alphaA, and alphaB:alphaB subunit interactions are similar. Taken together, these data suggest that the differences in the number of subunit contacts in the mixed assemblies give rise to the disproportionate dissociation of alphaB-crystallin subunits. Limited proteolysis mass spectrometry was also used to examine changes in protease accessibility during subunit exchange. The C-terminus of alphaA-crystallin was more susceptible to proteolytic attack in homo-oligomers than that of alphaB-crystallin. As subunit exchange proceeded, proteolysis of the alphaA-crystallin C-terminus increased, indicating that in the hetero-oligomeric form this tertiary motif is more exposed to solvent. These data were used to propose a refined arrangement for the interactions of the alpha-crystallin domains and C-terminal extensions of subunits within the alpha-crystallin assembly. In particular, we propose that the palindromic IPI motif of alphaB-crystallin gives rise to two orientations of the C-terminus.

Heat-induced structural transition of α-crystallin in the eye lens tissue observed by small-angle X-ray scattering

Heat-induced structural transitions of crystallins in the eye lens tissue have been studied by small-angle X-ray scattering. It was shown that a short-time (approximately 1 min) incubation of the bovine eye lens tissue at a temperature of about 60 degrees C leads to a pronounced shift of the small-angle X-ray diffraction maximum due to the short-range order of alpha-crystallin oligomers. This shift indicates an increase in the molecular mass of alpha-crystallin oligomers. The results are evidence that, in the native surrounding and at the native concentration of alpha-crystallin, heat-induced transition of alpha-crystallin quaternary structure takes place. Earlier, this transition of alpha-crystallin has been observed only in solutions and gels of this protein. The results confirm the identity of alpha-crystallin properties in vitro and in vivo.

Quaternary structure of alpha-crystallin is necessary for the binding of unfolded proteins: a surface plasmon resonance study.

The interactions between an oligomeric heat-shock protein, alpha-crystallin, and its individual subunits with unfolded proteins were monitored by surface plasmon resonance. Immobilization at the sensor chip allowed us for the first time to study isolated alpha-crystallin subunits under physiological conditions. We observe that these subunits, in contrast to alpha-crystallin oligomers, do not bind unfolded protein. Our data indicate that quaternary structure of alpha-crystallin is necessary for its chaperone-like activity.

Structural perturbation and enhancement of the chaperone-like activity of alpha-crystallin by arginine hydrochloride.

Structural perturbation of alpha-crystallin is shown to enhance its molecular chaperone-like activity in preventing aggregation of target proteins. We demonstrate that arginine, a biologically compatible molecule that is known to bind to the peptide backbone and negatively charged side-chains, increases the chaperone-like activity of calf eye lens alpha-crystallin as well as recombinant human alphaA- and alphaB-crystallins. Arginine-induced increase in the chaperone activity is more pronounced for alphaB-crystallin than for alphaA-crystallin. Other guanidinium compounds such as aminoguanidine hydrochloride and guanidine hydrochloride also show a similar effect, but to different extents. A point mutation, R120G, in alphaB-crystallin that is associated with desmin-related myopathy, results in a significant loss of chaperone-like activity. Arginine restores the activity of mutant protein to a considerable extent. We have investigated the effect of arginine on the structural changes of alpha-crystallin by circular dichroism, fluorescence, and glycerol gradient sedimentation. Far-UV CD spectra show no significant changes in secondary structure, whereas near-UV CD spectra show subtle changes in the presence of arginine. Glycerol gradient sedimentation shows a significant decrease in the size of alpha-crystallin oligomer in the presence of arginine. Increased exposure of hydrophobic surfaces of alpha-crystallin, as monitored by pyrene-solubilization and ANS-fluorescence, is observed in the presence of arginine. These results show that arginine brings about subtle changes in the tertiary structure and significant changes in the quaternary structure of alpha-crystallin and enhances its chaperone-like activity significantly. This study should prove useful in designing strategies to improve chaperone function for therapeutic applications.

Structural alterations of alpha-crystallin during its chaperone action.

The small heat-shock protein, alpha-crystallin, has chaperone ability whereby it stabilises proteins under stress conditions. In this study, alterations in the structure of alpha-crystallin during its interaction with a variety of substrate proteins (insulin, alpha-lactalbumin, ovotransferrin and serum albumin) under stress conditions have been examined using visible absorption, 31P-NMR and 1H-NMR and fluorescence spectroscopy. The fluorescence and 31P-NMR data imply that during the chaperone action of alpha-crystallin under reducing conditions, there is a slight increase in hydrophilicity of its N-terminal region and an alteration in flexibility of its C-terminal region, but overall, alpha-crystallin does not undergo a gross structural change. The fluorescence data suggest that substrate proteins interact with alpha-crystallin in a molten globule or intermediately folded state. The same conclusion is made from 1H-NMR spectroscopic monitoring of the interaction of alpha-crystallin with substrate proteins, e.g. the insulin B chain. The stoichiometry of interaction between alpha-crystallin and the various substrate proteins reveals that steric factors are important in determining the efficiency of interaction between the two proteins, i.e. on a molar subunit basis, alpha-crystallin is a more efficient chaperone protein with smaller substrate proteins. Comparison is also made between the high-molecular-mass (HMM) complexes formed between alpha-crystallin and ovotransferrin when reduced and heat stressed. Under heating conditions, fluorescence spectroscopy indicates that the HMM complex has a greater exposure of hydrophobicity to solution than that formed by reduction. Furthermore, in interacting with heated ovotransferrin, the C-terminal extension of the alphaB-crystallin subunit preferentially loses its flexibility suggesting that it is involved in stabilising bound ovotransferrin. By contrast, this extension is only partially reduced in flexibility in the HMM complex formed after reduction of ovotransferrin. The functional role of the C-terminal extensions in the chaperone action and the overall quaternary structure of alpha-crystallin is discussed.

Chaperone-like activity and quaternary structure of alpha-crystallin.

alpha-Crystallin has been shown to function as a molecular chaperone in preventing thermal aggregation of crystallins and other proteins. The molecular mechanism of this protection is not yet clear. gamma-Crystallin aggregates upon exposure to UV light. We have investigated the effect of the presence of alpha-crystallin in the photoaggregation process and find that alpha-crystallin does not prevent photoaggregation at low temperatures. The protection starts around 30 degrees C and steeply increases with temperature. The plot of protection ability versus temperature is sigmoidal, indicating a structural transition. Perturbation of the quaternary structure of alpha by non-thermal mode, such as 3 M urea, also results in enhanced protection. Pyrene, a hydrophobic fluorophore, is sparingly soluble in water. alpha-Crystallin enhances the solubility of pyrene by severalfold. Temperature dependence of this solubilization shows a transition around 30 degrees C (another at about 50 degrees C). Fluorescence intensity ratio of third and first peaks of pyrene emission (I3/I1,), indicative of hydrophobicity of the reporting area, also shows similar transitions suggesting enhanced hydrophobicity. Gel filtration experiments of irradiated samples indicate the complex formation between gamma- and alpha-crystallins. alpha-Crystallin does not prevent cold precipitation of gamma-crystallin. On the basis of these results, we hypothesize that alpha-crystallin prevents aggregation of non-native structures by providing appropriately placed hydrophobic surfaces. A structural transition above 30 degrees C enhances the protective ability, perhaps by increasing or reorganizing the hydrophobic surfaces. A similar temperature dependence has been reported for GroEL. Whether a structural switch, either activated by temperature, solvent conditions, or small molecule binding, forms a part of the general mechanism of chaperone activity needs to be investigated.

Structural properties of polydisperse biopolymer solutions: A light scattering study of bovine α‐crystallin

We have measured mean value of RHz, mean value of R2G1/2z, and mean value of Mw for individual fractions of the protein alpha-crystallin obtained by gel filtration of bovine lens nuclear extracts. A strong and monotonic decrease of mean value of RHz and mean value of Mw with increasing elution volume could be observed, indicating a broad size distribution. The experimental results are quantitatively consistent with a polymerization of monomeric units into linear chains, which may have a certain degree of flexibility. Using theoretical expressions for mean value of R2G and mean value of RH originally derived for semiflexible polymers in solution, we can self-consistently analyse the data from static and dynamic light scattering, and from electron microscopy experiments. We thus obtain detailed information on the molecular weight distribution and the quaternary structure of alpha-crystallin in these solutions.