Specificity of alphaA-crystallin binding to destabilized mutants of betaB1-crystallin

To elucidate the structural and energetic basis of attractive protein interactions in the aging lens, we investigated the binding of destabilized mutants of betaB1-crystallin to the lens chaperones, alpha-crystallins. We show that the mutations enhance the binding affinity to alphaA- but not alphaB-crystallin at physiological temperatures. Complex formation disrupts the dimer interface of betaB1-crystallin consistent with the binding of a monomer. Binding isotherms obtained at increasing concentrations of betaB1-crystallin deviate from a classic binding equilibrium and display cooperative-like behavior. In the context of betaB1-crystallin unfolding equilibrium, these characteristics are reflective of the concentration-dependent change in the population of a dimeric intermediate that has low affinity to alphaA-crystallin. In the lens, where alpha-crystallin binding sites are not regenerated, this may represent an added mechanism to maintain lens transparency.     

Association of partially folded lens betaB2-crystallins with the alpha-crystallin molecular chaperone

Age-related cataract is a result of crystallins, the predominant lens proteins, forming light-scattering aggregates. In the low protein turnover environment of the eye lens, the crystallins are susceptible to modifications that can reduce stability, increasing the probability of unfolding and aggregation events occurring. It is hypothesized that the alpha-crystallin molecular chaperone system recognizes and binds these proteins before they can form the light-scattering centres that result in cataract, thus maintaining the long-term transparency of the lens. In the present study, we investigated the unfolding and aggregation of (wild-type) human and calf betaB2-crystallins and the formation of a complex between alpha-crystallin and betaB2-crystallins under destabilizing conditions. Human and calf betaB2-crystallin unfold through a structurally similar pathway, but the increased stability of the C-terminal domain of human betaB2-crystallin relative to calf betaB2-crystallin results in the increased population of a partially folded intermediate during unfolding. This intermediate is aggregation-prone and prevents constructive refolding of human betaB2-crystallin, while calf betaB2-crystallin can refold with high efficiency. alpha-Crystallin can effectively chaperone both human and calf betaB2-crystallins from thermal aggregation, although chaperone-bound betaB2-crystallins are unable to refold once returned to native conditions. Ordered secondary structure is seen to increase in alpha-crystallin with elevated temperatures up to 60 degrees C; structure is rapidly lost at temperatures of 70 degrees C and above. Our experimental results combined with previously reported observations of alpha-crystallin quaternary structure have led us to propose a structural model of how activated alpha-crystallin chaperones unfolded betaB2-crystallin.     

Analysis of betaB1-crystallin unfolding equilibrium by spin and fluorescence labeling: evidence of a dimeric intermediate

A central step in understanding lens aging is to characterize the thermodynamic stability of its proteins and determine the consequences of changes in the primary sequence on their folding equilibria. For this purpose, destabilized mutations were introduced in betaB1-crystallin targeting the domain interface within the fold of a subunit. Global unfolding was monitored by tryptophan fluorescence while concomitant structural changes at the dimer interface were monitored by fluorescence and spin labels. Both spectral probes report explicit evidence of multi-state unfolding equilibrium. The biphasic nature of the unfolding curves was more pronounced at higher protein concentration. Distinct shifts in the midpoint of the second transition reflect the population of a dimeric intermediate. This intermediate may be a critical determinant for the life-long stability of the beta-crystallins and has important consequences on interactions with alpha-crystallin.     

Mechanism of chaperone function in small heat-shock proteins. Fluorescence studies of the conformations of T4 lysozyme bound to alphaB-crystallin

To further develop the mechanistic understanding of small heat-shock protein (sHSP) chaperone activity, we investigate the nature of the intermediate states recognized by alpha-crystallin and the conformations that are stably bound. The model substrates consist of a set of well characterized, destabilized T4 Lysozyme (T4L) mutants that have been shown to differentially bind alpha-crystallin in a manner that reflects their free-energy of unfolding. A new approach for the detection of complex formation is introduced based on the conformational sensitivity of the fluorescent probe bimane, site-specifically introduced in T4L. Emission spectra of bimane-labeled T4L reveal two distinct patterns of intensity changes upon binding that depend on the molar ratio of alpha-crystallin to T4L. This directly demonstrates the two-mode nature of the binding process by the alpha-crystallins. Biphasic binding isotherms, obtained and analyzed over a wide range of T4L concentrations, demonstrate a substantially quenched bimane fluorescence in the low affinity-bound T4L that is similar to the quenching level observed due to denaturant unfolding. Furthermore, the pattern of intensity changes that occur upon binding of a T4L variant, bimane-labeled at an alternative solvent-exposed site, establishes a direct correlation between the quenching level observed in binding and unfolding. The results can be interpreted in terms of a model where alpha-crystallin binds at least two conformationally distinct non-native states of T4L, one of which is substantially unfolded and is bound with low affinity. A high affinity binding mode to compact states may be relevant to chaperone function in the lens, where protein damage is unlikely to cause global unfolding.     

Chaperone activity of alpha-crystallins modulates intermediate filament assembly.

Intermediate filaments are generally regarded as one of the most insoluble and resilient cytoskeletal structures of eukaryotic cells. In extracts from the ocular lens, we noticed an unusually high level of vimentin in a soluble, non-filamentous form. Immunoprecipitation of this soluble vimentin resulted in the co-precipitation of alpha-crystallins. The alpha-crystallins are homologous to the small heat shock proteins (sHSPs) and have recently been identified as molecular chaperones, capable of preventing the heat-induced aggregation of proteins. We find that the alpha-crystallins dramatically inhibit the in vitro assembly of GFAP and vimentin in an ATP-independent manner. This inhibition is also independent of the phosphorylation state of the alpha-crystallin polypeptides and each one of the four polypeptides, either alpha A1-, alpha A2-, alpha B1- or alpha B2-crystallin, are equally effective in this inhibition. Furthermore, we show that alpha-crystallins can increase the soluble pool of GFAP when added to preformed filaments. Electron microscopy demonstrated that alpha-crystallin particles could bind to intermediate filaments in a regular fashion, the spacing coinciding with the molecular length of GFAP. This is the first report, as far as we are aware, of a chaperone being involved in intermediate filament assembly and implicates chaperones in the remodeling of intermediate filaments during development and cell differentiation.     

In vivo heteromer formation. Expression of soluble betaA4-crystallin requires coexpression of a heteromeric partner

The beta-crystallins are a family of long-lived, abundant structural proteins that are coexpressed in the vertebrate lens. As beta-crystallins form heteromers, a process that involves transient exposure of hydrophobic interfaces, we have examined whether in vivobeta-crystallin assembly is enhanced by protein chaperones, either small heat shock proteins, Hsp27 or alphaB-crystallin, or Hsp70. We show here that betaA4-crystallin is abundantly expressed in HeLa cells, but rapidly degraded, irrespective of the presence of Hsp27, alphaB-crystallin or Hsp70. Degradation is even enhanced by Hsp70. Coexpression of betaA4-crystallin with betaB2-crystallin yielded abundant soluble betaA4-betaB2-crystallin heteromers; betaB1-crystallin was much less effective in solubilizing betaA4-crystallin. As betaB2-crystallin competed for betaA4-crystallin with Hsp70 and the proteasomal degradation pathway, betaB2-crystallin probably captures an unstable betaA4-crystallin intermediate. We suggest that the proper folding of betaA4-crystallin is not mediated by general chaperones but requires a heteromeric partner, which then also acts as a dedicated chaperone towards betaA4-crystallin.     

Deamidation in human lens betaB2-crystallin destabilizes the dimer

Two major determinants of the transparency of the lens are protein-protein interactions and stability of the crystallins, the structural proteins in the lens. betaB2 is the most abundant beta-crystallin in the human lens and is important in formation of the complex interactions of lens crystallins. betaB2 readily forms a homodimer in vitro, with interacting residues across the monomer-monomer interface conserved among beta-crystallins. Due to their long life spans, crystallins undergo an unusually large number of modifications, with deamidation being a major factor. In this study the effects of two potential deamidation sites at the monomer-monomer interface on dimer formation and stability were determined. Glutamic acid substitutions were constructed to mimic the effects of previously reported deamidations at Q162 in the C-terminal domain and at Q70, its N-terminal homologue. The mutants had a nativelike secondary structure similar to that of wild type betaB2 with differences in tertiary structure for the double mutant, Q70E/Q162E. Multiangle light scattering and quasi-elastic light scattering experiments showed that dimer formation was not interrupted. In contrast, equilibrium unfolding and refolding in urea showed destabilization of the mutants, with an inflection in the transition of unfolding for the double mutant suggesting a distinct intermediate. These results suggest that deamidation at critical sites destabilizes betaB2 and may disrupt the function of betaB2 in the lens.     

Mutation of interfaces in domain-swapped human betaB2-crystallin

The superfamily of eye lens betagamma-crystallins is highly modularized, with Greek key motifs being used to form symmetric domains. Sequences of monomeric gamma-crystallins and oligomeric beta-crystallins fold into two domains that pair about a further conserved symmetric interface. Conservation of this assembly interface by domain swapping is the device adopted by family member betaB2-crystallin to form a solution dimer. However, the betaB1-crystallin solution dimer is formed from an interface used by the domain-swapped dimer to form a tetramer in the crystal lattice. Comparison of these two structures indicated an intriguing relationship between linker conformation, interface ion pair networks, and higher assembly. Here the X-ray structure of recombinant human betaB2-crystallin showed that domain swapping was determined by the sequence and not assembly conditions. The solution characteristics of mutants that were designed to alter an ion pair network at a higher assembly interface and a mutant that changed a proline showed they remained dimeric. X-ray crystallography showed that the dimeric mutants did not reverse domain swapping. Thus, the sequence of betaB2-crystallin appears well optimized for domain swapping. However, a charge-reversal mutation to the conserved domain-pairing interface showed drastic changes to solution behavior. It appears that the higher assembly of the betagamma-crystallin domains has exploited symmetry to create diversity while avoiding aggregation. These are desirable attributes for proteins that have to exist at very high concentration for a very long time.     

The function of alpha-crystallin in vision

The alpha-crystallins account for approximately one-third of the total soluble protein in the lens, contributing to its refractive power. In addition, alpha-crystallin also has a chaperone-like function and thus can bind unfolding lens proteins. Alpha B-crystallin is also found outside the lens, having an extensive tissue distribution. It is over-expressed in response to stresses of all kinds, where it is thought to serve a general protective function. Recently, it has been shown in humans that naturally occurring point mutations in the alpha-crystallins result in a deficit in chaperone-like function, and cause cataracts as well as a desmin-related myopathy. This review summarizes much of the past and current knowledge concerning the structure and functions of alpha-crystallin.     

Formation of betaA3/betaB2-crystallin mixed complexes: involvement of N- and C-terminal extensions.

The sequence extensions of the beta-crystallin subunits have been suggested to play an important role in the oligomerization of these eye lens proteins. This, in turn, may contribute to maintaining lens transparency and proper light refraction. In homo-dimers of the betaA3- and betaB2-crystallin subunits, these extensions have been shown by (1)H-NMR spectroscopy to be solvent-exposed and highly flexible. In this study, we show that betaA3- and betaB2-crystallins spontaneously form mixed betaA3/betaB2-crystallin complexes, which, from analytical ultracentrifugation experiments, are dimeric at low concentrations (<1 mg ml(-1)) and tetrameric at higher protein concentrations. (1)H-NMR spectroscopy reveals that in the betaA3/betaB2-crystallin tetramer, the N-terminal extensions of betaA3-crystallin remain water-exposed and flexible, whereas both N- and C-terminal extensions of betaB2-crystallin lose their flexibility. We conclude that both extensions of betaB2-crystallin are involved in protein-protein interactions in the betaA3/betaB2-crystallin hetero-tetramer. The extensions may stabilize and perhaps promote the formation of this mixed complex.     

Unfolding crystallins: the destabilizing role of a beta-hairpin cysteine in betaB2-crystallin by simulation and experiment

The thermodynamic and kinetic stabilities of the eye lens family of betagamma-crystallins are important factors in the etiology of senile cataract. They control the chance of proteins unfolding, which can lead to aggregation and loss of transparency. betaB2-Crystallin orthologs are of low stability and comprise two typical betagamma-crystallin domains, although, uniquely, the N-terminal domain has a cysteine in one of the conserved folded beta-hairpins. Using high-temperature (500 K) molecular dynamics simulations with explicit solvent on the N-terminal domain of rodent betaB2-crystallin, we have identified in silico local flexibility in this folded beta-hairpin. We have shown in vitro using two-domain human betaB2-crystallin that replacement of this cysteine with a more usual aromatic residue (phenylalanine) results in a gain in conformational stability and a reduction in the rate of unfolding. We have used principal components analysis to visualize and cluster the coordinates from eight separate simulated unfolding trajectories of both the wild-type and the C50F mutant N-terminal domains. These data, representing fluctuations around the native well, show that although the mutant and wild-type appear to behave similarly over the early time period, the wild type appears to explore a different region of conformational space. It is proposed that the advantage of having this low-stability cysteine may be correlated with a subunit-exchange mechanism that allows betaB2-crystallin to interact with a range of other beta-crystallin subunits.     

Human lens high-molecular-weight alpha-crystallin aggregates

Alpha-crystallin high-molecular-weight (HMW) aggregates can be formed in vitro by many mechanisms, but the mechanism of in vivo aggregation has not been clearly established. HMW and LMW (low-molecular-weight) alpha-crystallins were isolated from human lenses 50-60 years of age and some spectroscopic measurements were performed. Conformational differences were suggested based on data of increased bis-ANS (4,4'-dianilino-1,1'-binaphthalene-5, 5'-disulfonic acid) and ThT (thioflavin T) fluorescence as well as increased far-UV and decreased near-UV circular dichroism (CD). These results indicated that HMW alpha-crystallin was more hydrophobic than LMW alpha-crystallin, possibly resulting from partial unfolding of alpha-crystallin. On the other hand, the increased ThT fluorescence and far-UV CD intensities indicate that an increased amount of beta-sheet conformation was involved in aggregation. These data, along with little difference in chaperone-like activity between the LMW and HMW alpha-crystallins, strongly suggest that HMW alpha-crystallin aggregates resulted from partial unfolding and disassembling-reassembling of LMW alpha-crystallin caused by posttranslational modification rather than chaperone complex formation.     

Cataract-linked γD-crystallin mutants have weak affinity to lens chaperones α-crystallins

To test the hypothesis that α-crystallin chaperone activity plays a central role in maintenance of lens transparency, we investigated its interactions with γ-crystallin mutants that cause congenital cataract in mouse models. Although the two substitutions, I4F and V76D, stabilize a partially unfolded γD-crystallin intermediate, their affinities to α-crystallin are marginal even at relatively high concentrations. Detectable binding required further reduction of γD-crystallin stability which was achieved by combining the two mutations. Our results demonstrate that mutants and possibly age-damaged γ-crystallin can escape quality control by lens chaperones rationalizing the observation that they nucleate protein aggregation and lead to cataract.     

Association properties of betaB1- and betaA3-crystallins: ability to form heterotetramers

As major constituents of the mammalian lens, beta-crystallins associate into dimers, tetramers, and higher-order complexes to maintain lens transparency and refractivity. A previous study has shown that dimerization of betaB2- and betaA3-crystallins is energetically highly favored and entropically driven. While heterodimers further associate into higher-order complexes in vivo, a significant level of reversibly associated tetrameric crystallin has not been previously observed in vitro. To enhance our understanding of the interactions between beta-crystallins, we characterized the association of betaB1-crystallin, a major component of large beta-crystallin complexes (beta-high), with itself and with betaA3-crystallin. Mouse betaB1-crystallin and human betaA3-crystallin were expressed in Escherichia coli and purified chromatographically. Their association was then characterized using size-exclusion chromatography, native gel electrophoresis, isoelectric focusing, and analytical sedimentation equilibrium centrifugation. When present alone, each beta-crystallin associates into homodimers; however, no tetramer formation is seen. Once mixing has taken place, formation of a heterocomplex between betaB1- and betaA3-crystallins is observed using size-exclusion chromatography, native gel electrophoresis, isoelectric focusing, and sedimentation equilibrium. In contrast to results previously obtained after betaB2- and betaA3-crystallins had been mixed, mixed betaB1- and betaA3-crystallins show a dimer-tetramer equilibrium with a K d of 1.1 muM, indicating that these two beta-crystallins associate predominantly into heterotetramers in vitro. Thus, while each purified beta-crystallin associates only into homodimers and under the conditions studied mixed betaB2- and betaA3-crystallins form a mixture of homo- and heterodimers, mixed betaB1- and betaA3-crystallins associate predominantly into heterotetramers in equilibrium with heterodimers. These findings suggest a unique role for betaB1-crystallin in promoting higher-order crystallin association in the lens.     

Identification and properties of anti-chaperone-like peptides derived from oxidized bovine lens betaL-crystallins

Thermal aggregation of betaL-crystallin was higher in the presence of peptide fragments generated from oxidized and trypsin-digested betaL-crystallin compared with thermal aggregation of the control proteins without oxidized betaL-crystallin fragments. Increased aggregation of betaL-crystallin was also observed despite the presence of alpha-crystallin (which has anti-aggregating properties) in the system. Self-aggregation of the oxidized betaL-crystallin fragments per se was not observed under the experimental conditions. Reverse-phase HPLC analysis of the precipitate obtained after heating a mixture of betaL-crystallin and oxidized betaL-crystallin fragments revealed that more than one peptide co-precipitates with betaL-crystallin. Electrospray mass spectrometry analysis of the peptides revealed that the molecular weight(s) of the peptides ranged from 1400-1800. Tandem mass spectrometry and a data base search revealed that two of the peptides originated from betaA4-crystallin (LTIFEQENFLGR, residues 121-132) and betaB3-crystallin (AINGTWVGYEFPGYR, residues 153-167) respectively. Oxidized synthetic peptides representing the same sequence were also found to enhance the aggregation of betaL-crystallin in a manner similar to oxidized lens betaL-crystallin peptides. These data suggest that the polypeptides generated after oxidation and proteolysis of betaL-crystallins interact with denaturing proteins and facilitate their aggregation and light scattering, thus behaving like anti-chaperones.     

Protein-protein interactions among human lens acidic and basic beta-crystallins

Human lens beta-crystallin contains four acidic (betaA1-->betaA4) and three basic (betaB1-->betaB3) subunits. They oligomerize in the lens, but it is uncertain which subunits are involved in the oligomerization. We used a two-hybrid system to detect protein-protein interactions systematically. Proteins were also expressed for some physicochemical studies. The results indicate that all acidic-basic pairs (betaA-betaB) except betaA4-betaBs pairs show strong hetero-molecular interactions. For acidic or basic pairs, only two pairs (betaA1-betaA1 and betaA3-betaA3) show strong self-association. betaA2 and betaA4 show very weak self-association, which arises from their low solubility. Confocal fluorescence microscopy shows enormous protein aggregates in betaA2- or betaA4-crystallin transfected cells. However, coexpression with betaB2-crystallin decreased both the number and size of aggregates. Circular dichroism indicates subtle differences in conformation among beta-crystallins that may have contributed to the differences in interactions.     

Human lens beta-crystallin solubility

The human lens is composed primarily of water and proteins called crystallins. Insolubility of these crystallins is correlated with aging and cataractogenesis. The alpha-crystallins have chaperone-like activity in maintaining the solubility of denatured beta- and gamma-crystallins. One established test of this chaperone activity is the ability of alpha-crystallin to prevent thermal destabilization of beta-crystallins. Several studies have addressed the effects of structural modifications of alpha-crystallin on chaperone activity, but little is known about the solubilities of the various beta-crystallins or the effects of post-translational modifications. Understanding the solubilities of different forms of beta-crystallins is important to elucidating the mechanism of chaperone activity. In this study, the solubilities of beta-crystallins were examined. The beta-crystallins included the gene products of betaB2, betaA1/A3, betaA4, and betaB1 as well as forms modified in vivo. Analysis of the beta-crystallins by high performance liquid chromatography and mass spectrometry before and after heating revealed large differences in the relative solubilities of the beta-crystallins. These results demonstrate a decreased solubility of specific beta-crystallins and post-translational modifications that may play a role in the crystallin insolubility associated with aging and cataract.     

Energetics of domain-domain interactions and entropy driven association of beta-crystallins

Beta-crystallins are major protein constituents of the mammalian lens, where their stability and association into higher order complexes are critical for lens clarity and refraction. They undergo modification as the lens ages, including cleavage of their terminal extensions. The energetics of betaA3- and betaB2-crystallin association was studied using site-directed mutagenesis and analytical ultracentrifugation. Recombinant (r) murine wild type betaA3- and betaB2-crystallins were modified by removal of either the N-terminal extension of betaA3 (rbetaA3Ntr) or betaB2 (rbetaB2Ntr), or both the N- and C-terminal extensions of betaB2 (rbetaB2NCtr). The proteins were expressed in Sf9 insect cells or Escherichia coli and purified by gel-filtration and ion-exchange chromatography. All beta-crystallins studied demonstrated fast reversible monomer-dimer equilibria over the temperature range studied (5-35 degrees C) with a tendency to form tighter dimers at higher temperatures. The N-terminal deletion of rbetaA3 (rbetaA3Ntr) significantly increases the enthalpy (+10.9 kcal/mol) and entropy (+40.7 cal/deg mol) of binding relative to unmodified protein. Removal of both N- and C-terminal extensions of rbetaB2 also increases these parameters but to a lesser degree. Deletion of the betaB2-crystallin N-terminal extension alone (rbetaB2Ntr) gave almost no change relative to rbetaB2. The resultant net negative changes in the binding energy suggest that betaAlpha3- and betaB2-crystallin association is entropically driven. The thermodynamic consequences of the loss of betaAlpha3-crystallin terminal extensions by in vivo proteolytic processing could increase their tendency to associate and so promote the formation of higher order associates in the aging and cataractous lens.     

The benefits of being β-crystallin heteromers: βB1-crystallin protects βA3-crystallin against aggregation during co-refolding

β-Crystallins are the major structural proteins in mammalian lens, and their stability is critical in maintaining the transparency and refraction index of the lens. Among the seven β-crystallins, βA3-crystallin and βB1-crystallin, an acidic and a basic β-crystallin, respectively, can form heteromers in vivo. However, the physiological roles of the heteromer have not been fully elucidated. In this research, we studied whether the basic β-crystallin facilitates the folding of acidic β-crystallin. Equilibrium folding studies revealed that the βA3-crystallin and βB1-crystallin homomers and the βA3/βB1-crystallin heteromer all undergo similar five-state folding pathways which include one dimeric and two monomeric intermediates. βA3-Crystallin was found to be the most unstable among the three proteins, and the transition curve of βA3/βB1-crystallin was close to that of βB1-crystallin. The dimeric intermediate may be a critical determinant in the aggregation process and thus is crucial to the lifelong stability of the β-crystallins. A comparison of the Gibbs free energy of the equilibrium folding suggested that the formation of heteromer contributed to the stabilization of the dimer interface. On the other hand, βA3-crystallin, the only protein whose refolding is challenged by serious aggregation, can be protected by βB1-crystallin in a dose-dependent manner during the kinetic co-refolding. However, the protection is not observed in the presence of the pre-existed well-folded βB1-crystallin. These findings suggested that the formation of β-crystallin heteromers not only stabilizes the unstable acidic β-crystallin but also protects them against aggregation during refolding from the stress-denatured states.     

Unfolding of human lens recombinant betaB2- and gammaC-crystallins

betaB2- and gammaC-crystallins belong to the betagamma-crystallin superfamily and have very similar structures. Molecular spectroscopic techniques such as UV-visible absorption, circular dichroism, and fluorescence indicate they have similar biophysical properties. Their structures are characterized by the presence of two domains consisting of four Greek key motifs. The only difference is the connecting peptide of the two domains, which is flexible in gamma-crystallin but extended in beta-crystallin; thus, an intradomain association and a monomer are formed in gamma-crystallin and an interdomain association and a dimer are formed in beta-crystallin. The difference may be reflected in the thermodynamic stability. In the present study, we calculated the standard free-energy by equilibrium unfolding transition in guanidine hydrochloride using three spectroscopic parameters: absorbance at 235nm, Trp fluorescence intensity at 320nm, and far-UV circular dichroism at 223nm. Global analyses indicate that both dimeric betaB2- and monomeric gammaC-crystallins are a better fit to a three-state model than to a two-state model. In terms of standard free-energy, deltaG(0)(H(2)O,i) both betaB2-crystallin and gammaC-crystallin are stable proteins and dimeric betaB2-crystallin is more stable than the monomeric gammaC-crystallin. The significance of the thermodynamic stability for betaB2- and gammaC-crystallins may be related to their functions in the lens.