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.     

Monomer-dimer equilibrium of normal and modified beta A3-crystallins: experimental determination and molecular modeling

Beta- and gamma-crystallins are major protein constituents of the mammalian lens, where their stability and association into higher order complexes are critical for clarity and refraction. Two regions of the betagamma-crystallins have been suggested to modulate protein association, namely, the flexible N-terminal extensions and the intramolecular domain interfaces. The oligomeric state of wild-type recombinant murine betaA3-crystallin (rbetaA3) was compared to that of modified betaA3-crystallins with either an N-terminal deletion of residues 1 to 29 (rbetaA3tr) or with residues 114 to 123 of the interdomain linker replaced with the analogous linker from murine gammaB-crystallin (rbetaA3cp). All three proteins exhibited reversible monomer-dimer formation. The modifications to the N-terminus and domain linker resulted in tighter dimer formation as compared to wild-type protein as indicated by disassociation constants determined by sedimentation equilibrium: 6.62 x 10(-6) M (rbetaA3), 0.86 x 10(-6) M (rbetaA3cp), and 1.83 x 10(-7) M (rbetaA3tr). Homology modeling of betaA3-crystallins and solvation energy calculations also predicted tighter binding of the modified crystallins consistent with the centrifugation results. The findings suggest that under physiological conditions betaA3 crystallin exists in a dynamic equilibrium between monomeric and dimeric protein and that modification, especially to the N-terminal extension, can promote self-association.     

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.     

BetaB2-crystallin undergoes extensive truncation during aging in human lenses

Based on the present literature, it is unclear whether betaB2-crystallin undergoes age-related truncation in human lenses. To answer this question, the purpose of this study was to determine in vivo truncation of betaB2-crystallin in human lenses during aging by examining its fragments in the beta(H)-crystallin fraction. The WS-protein fraction was isolated from lenses of desired ages and separated by a size-exclusion Agarose A 1.5m column to recover alpha-, beta(H)-, beta(L)-, and gamma-crystallin fractions. The beta(H)-crystallin fractions, isolated from lenses of 24- and 70-year-old donors, were utilized for two-dimensional (2D) gel electrophoresis (isoelectric focusing in the first dimension followed by SDS-PAGE in the second dimension). The partial N-terminal sequences of the desired fragments (Molecular weights [M(r)]<18-19kDa) from a 2D-gel of WS-proteins from lenses of a 70-year-old donor were determined. More than 37 crystallin fragments with M(r) between 4 and 19kDa were observed on a 2D-gel. Nine fragments in beta(H)-crystallin fraction were from betaB2-crystallin but additional single fragments of alphaA-, gammas-, betaA4, and of either gammaB-, gammaC- or gammaD-crystallins were also observed. Seven cleavage sites in the betaB2-crystallin were identified, which included two sites at Q(7)-A(8) and A(8)-G(9) bonds in the N-terminal extension, two sites at E(46)-K(47) and G(49)-S(50) bonds in the motif 1, one site at S(94) -S(95) in the motif 2, and two sites at N(115)-F(116) and Q(135)-Y(136) in motif 3. No fragments with cleavage in the motif 4 and C-terminal extension of betaB2-crystallin were seen. Apparently, three betaB2-crystallin fragments with only N-terminal cleavage and five with both N- and C-terminal cleavages were observed. Additional fragments with cleavage sites at Q(54)-Y(55) in alphaA-crystallin, at E(112)-N(113) in betaA4-crystallin, at G(4)-T(5) in gammas-crystallin, at M(69)-G(70) in either gammaB-, gammaC- or gammaD-crystallins (three have identical sequences at the cleaved bond), and at G(1)-K(2) in gammaB or gammaC (both have identical sequences at the cleavage site) were observed.Conclusions. The results showed that betaB2-crystallin undergoes age-related truncation producing fragments with M(r) between 4 and 19kDa that existed in the beta(H)-crystallin oligomer. The beta(H)-crystallin fraction also contained single fragments of alpha-, betaA4-, gammas-, and other gamma-crystallins.     

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.     

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.     

Calcium-binding to lens betaB2- and betaA3-crystallins suggests that all beta-crystallins are calcium-binding proteins

Crystallins are the major proteins of a mammalian eye lens. The topologically similar eye lens proteins, beta- and gamma-crystallins, are the prototype and founding members of the betagamma-crystallin superfamily. Betagamma-crystallins have until recently been regarded as structural proteins. However, the calcium-binding properties of a few members and the potential role of betagamma-crystallins in fertility are being investigated. Because the calcium-binding elements of other member proteins, such as spherulin 3a, are not present in betaB2-crystallin and other betagamma-crystallins from fish and mammalian genomes, it was argued that lens betagamma-crystallins should not bind calcium. In order to probe whether beta-crystallins can bind calcium, we selected one basic (betaB2) and one acidic (betaA3) beta-crystallin for calcium-binding studies. Using calcium-binding assays such as 45Ca overlay, terbium binding, Stains-All and isothermal titration calorimetry, we established that both betaB2- and betaA3-crystallin bind calcium with moderate affinity. There was no significant change in their conformation upon binding calcium as monitored by fluorescence and circular dichroism spectroscopy. However, 15N-1H heteronuclear single quantum correlation NMR spectroscopy revealed that amide environment of several residues underwent changes indicating calcium ligation. With the corroboration of calcium-binding to betaB2- and betaA3-crystallins, we suggest that all beta-crystallins bind calcium. Our results have important implications for understanding the calcium-related cataractogenesis and maintenance of ionic homeostasis in the lens.     

Truncation of motifs III and IV in human lens betaA3-crystallin destabilizes the structure

The purpose of our study was to determine the effects of specific truncations on the structural properties of human betaA3-crystallin. The following eight deletion mutants of betaA3-crystallin were generated: (i) N-terminal extension (NTE) 21 amino acids (betaA3[21] mutant), (ii) NTE 22 amino acids (betaA3[22] mutant), (iii) NTE (betaA3[N] mutant), (iv) NTE plus motif I (betaA3[N+I] mutant), (v) NTE plus motifs I and II (betaA3[N+I+II] mutant), (vi) NTE plus motifs I and II and connecting peptide (betaA3[N+I+II+CP] mutant), (vii) motifs III and IV (betaA3[III+IV] mutant), and (viii) motif IV (betaA3 [IV] mutant). The DNA sequencing and MALDI-TOF mass spectrometric methods confirmed desired specific deletions, and the purified mutant proteins exhibited a single band during SDS-PAGE analysis. When ANS bound, all the mutant proteins exhibited fluorescence quenching and a red shift, suggesting that the truncations caused changes in the exposed hydrophobic patches. The CD spectra showed that deletion of either NTE or the N-terminal domain (motifs I and II) had a relatively weaker effect on the structural stability than deletion of the C-terminal domain (motifs III and IV). Intrinsic Trp fluorescence spectral studies suggested changes in the microenvironment of the mutant proteins following truncations. HPLC multiangle light scattering analyses showed that truncation led to higher-order aggregation compared to that in the wild-type protein. Equilibrium unfolding and refolding of WT betaA3 with urea were best fit to a three-state model with transition midpoints at 2.2 and 3.1 M urea. However, the two transition midpoints of betaA3[21] and betaA3[22] and betaA3[N] mutants were similar to those of the wild type, suggesting that these truncations had a minimal effect on structural stabilization. Further, the mutant proteins containing the N-terminal domain (i.e., betaA3[III+IV] and betaA3[IV] mutants) exhibited higher transition midpoints compared to the transition midpoints of the mutant protein with the C-terminal domain (i.e., betaA3[N+I+II+CP] mutant). The results suggested that the N-terminal domain is relatively more stable than the C-terminal domain in betaA3-crystallin.     

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.     

Deamidation destabilizes and triggers aggregation of a lens protein, betaA3-crystallin

Protein aggregation is a hallmark of several neurodegenerative diseases and also of cataracts. The major proteins in the lens of the eye are crystallins, which accumulate throughout life and are extensively modified. Deamidation is the major modification in the lens during aging and cataracts. Among the crystallins, the betaA3-subunit has been found to have multiple sites of deamidation associated with the insoluble proteins in vivo. Several sites were predicted to be exposed on the surface of betaA3 and were investigated in this study. Deamidation was mimicked by site-directed mutagenesis at Q42 and N54 on the N-terminal domain, N133 and N155 on the C-terminal domain, and N120 in the peptide connecting the domains. Deamidation altered the tertiary structure without disrupting the secondary structure or the dimer formation of betaA3. Deamidations in the C-terminal domain and in the connecting peptide decreased stability to a greater extent than deamidations in the N-terminal domain. Deamidation at N54 and N155 also disrupted the association with the betaB1-subunit. Sedimentation velocity experiments integrated with high-resolution analysis detected soluble aggregates at 15%-20% in all deamidated proteins, but not in wild-type betaA3. These aggregates had elevated frictional ratios, suggesting that they were elongated. The detection of aggregates in vitro strongly suggests that deamidation may contribute to protein aggregation in the lens. A potential mechanism may include decreased stability and/or altered interactions with other beta-subunits. Understanding the role of deamidation in the long-lived crystallins has important implications in other aggregation diseases.     

Characterization of a sodium deoxycholate-activatable proteinase activity associated with betaA3/A1-crystallin of human lenses.

A human lens proteinase was purified by a five-step procedure that included two consecutive size-exclusion agarose A 1.5 m chromatographies, a preparative non-denaturing gel-electrophoretic separation, HPLC on a size-exclusion column (TSK G-3000 PW(XL)) followed by preparative isoelectric focusing. A 2300-fold purified enzyme showed a major band of 22 kDa during SDS-PAGE, a pH optimum of 7.8, pI between 4.5 and 5.0, a loss of activity above 45 degrees C and a serine type nature. The partial N-terminal sequence of the enzyme, i.e. P-M-P-G-S-L-G-P-W, matched with the sequence of human lens betaA3/A1-crystallin starting at residue No. 23. Based on the Western blot results of the enzyme with five different site-specific polyclonal antibodies raised against betaA3/A1-crystallin, it was concluded that the 22 kDa crystallin enzyme had a cleaved N-terminus but an intact C-terminus. The betaA3/A1-crystallin, isolated from human lenses, also exhibited proteinase activity following detergent activation and size-exclusion chromatography. The mouse recombinant betaA3/A1-crystallin proteinase was purified by the above five-step procedure, from a homogenate of Sf-9 cells transfected with baculovirus containing the full length coding sequence of betaA3/A1-crystallin. The mouse 22 kDa species also exhibited proteinase activity and immunoreactivity with anti-betaA3/A1-C-terminal antibody. Together, the data suggest that a truncated species of betaA3/A1-crystallin exhibits proteinase activity.     

Age-related degradation of betaA3/A1-crystallin in human lenses.

The aim of this study was to determine age-related degradation of betaA3/A1-crystallin in human lenses. The betaA3/A1-crystallin fragments were identified by Western blot analysis using two site-specific anti-betaA3/A1-crystallin antibodies. The first antibody was raised against a N-terminal region (residues 35-66), and the second to the C-terminal (residues 203-214) region of the crystallin. During the analyses, either preparative SDS-PAGE-separated fragments from betaH-crystallin fraction or water-soluble (WS) protein fractions from lenses of different aged donors were used. In lenses from 27- to 30-year-old donors, four major crystallin fragments of about 5, 16, 17, and 18 kDa immunoreacted with the anti-betaA3/A1-N-terminal antibody, suggesting their intact N-terminus but cleaved C-terminus. A similar analysis with the anti-betaA3/A1-C-terminal antibody identified 15-, 18-, 19-, and 20-kDa species and also five species between 4 and 11 kDa that had intact C-terminus but cleaved N-terminus. In lenses from a 5-year-old donor only two crystallin species, a major 15-kDa and a minor 18-kDa species, showed an intact N-terminus and cleaved C-terminus, whereas, eight species with Mr's between 4 and 19 kDa exhibited intact C-terminus but cleaved N-terminus. Upon two-dimensional gel electrophoresis of a betaH-crystallin fraction from the lenses of a 70-year-old donor, a degradation profile almost similar to the crystallin mentioned above was observed. However, the existence of multiple spots with identical Mr's of truncated betaA3/A1-crystallin species on the 2D-gel suggests their existence as isoforms (identical size species with different charges) because of post-translational modifications. Five species of 4, 6, 11, 15, and 18 kDa showed an identical partial N-terminal sequence of N-F-Q-G, suggesting cleavage at the E39-N40 bond during their production. Together, the data suggest that the majority of age-related cleavages in betaA3/A1-crystallin occur at the N-terminal region, with a major cleavage site at the E39-N40 bond generating some of these fragments.     

A nonsense mutation in CRYBB1 associated with autosomal dominant cataract linked to human chromosome 22q

Autosomal dominant cataract is a clinically and genetically heterogeneous lens disorder that usually presents as a sight-threatening trait in childhood. Here we have mapped dominant pulverulent cataract to the beta-crystallin gene cluster on chromosome 22q11.2. Suggestive evidence of linkage was detected at markers D22S1167 (LOD score [Z] 2.09 at recombination fraction [theta] 0) and D22S1154 (Z=1.39 at theta=0), which closely flank the genes for betaB1-crystallin (CRYBB1) and betaA4-crystallin (CRYBA4). Sequencing failed to detect any nucleotide changes in CRYBA4; however, a G-->T transversion in exon 6 of CRYBB1 was found to cosegregate with cataract in the family. This single-nucleotide change was predicted to introduce a translation stop codon at glycine 220 (G220X). Expression of recombinant human betaB1-crystallin in bacteria showed that the truncated G220X mutant was significantly less soluble than wild type. This study has identified the first CRYBB1 mutation associated with autosomal dominant cataract in humans.     

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.     

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.     

Crystal structure of truncated human betaB1-crystallin

Crystallins are long-lived proteins packed inside eye lens fiber cells that are essential in maintaining the transparency and refractive power of the eye lens. Members of the two-domain betagamma-crystallin family assemble into an array of oligomer sizes, forming intricate higher-order networks in the lens cell. Here we describe the 1.4 angstroms resolution crystal structure of a truncated version of human betaB1 that resembles an in vivo age-related truncation. The structure shows that unlike its close homolog, betaB2-crystallin, the homodimer is not domain swapped, but its domains are paired intramolecularly, as in more distantly related monomeric gamma-crystallins. However, the four-domain dimer resembles one half of the crystallographic bovine betaB2 tetramer and is similar to the engineered circular permuted rat betaB2. The crystal structure shows that the truncated betaB1 dimer is extremely well suited to form higher-order lattice interactions using its hydrophobic surface patches, linker regions, and sequence extensions.     

Regions Outside the α-Crystallin Domain of the Small Heat Shock Protein Hsp26 Are Required for Its Dimerization

Small heat shock proteins (sHsps) are a ubiquitous family of molecular chaperones. They form homo-oligomers, composed of mostly 24 subunits. The immunoglobulin-like α-crystallin domain, which is flanked by N- and C-terminal extensions, is the most conserved element in sHsps. It is assumed to be the dimeric building block from which the sHsp oligomers are assembled.Hsp26 from Saccharomyces cerevisiae is a well-characterized member of this family. With a view to study the structural stability and oligomerization properties of its α-crystallin domain, we produced a series of α-crystallin domain constructs. We show that a minimal α-crystallin domain can, against common belief, be monomeric and stably folded. Elongating either the N- or the C-terminus of this minimal α-crystallin domain with the authentic extensions leads to the formation of dimeric species. In the case of N-terminal extensions, their population is dependent on the presence of the complete so-called Hsp26 “middle domain”. For the C-terminal extensions, the presence of the conserved IXI motif of sHsps is necessary and sufficient to induce dimerization, which can be inhibited by increasing ionic strength. Dimerization does not induce major changes in secondary structure of the Hsp26 α-crystallin domain. A thermodynamic analysis of the monomeric and dimeric constructs revealed that dimers are not significantly stabilized against thermal and chemical denaturation in comparison to monomers, supporting our notion that dimerization is not a prerequisite for the formation of a well-folded Hsp26 α-crystallin domain.     

Dimerization of beta B2-crystallin: the role of the linker peptide and the N- and C-terminal extensions.

beta B2- and gamma B-crystallins of vertebrate eye lens are 2-domain proteins in which each domain consists of 2 Greek key motifs connected by a linker peptide. Although the folding topologies of beta B2- and gamma B-domains are very similar, gamma B-crystallin is always monomeric, whereas beta B2-crystallin associates to homodimers. It has been suggested that the linker or the protruding N- and C-terminal arms of beta B2-crystallin (not present in gamma B) are a necessary requirement for this association. In order to investigate the role of these segments for dimerization, we constructed two beta B2 mutants. In the first mutant, the linker peptide was replaced with the one from gamma B (beta B2 gamma L). In the second mutant, the N- and C-terminal arms of 15- and 12-residues length were deleted (beta B2 delta NC). The beta B2 gamma L mutant is monomeric, whereas the beta B2 delta NC mutant forms dimers and tetramers that cannot be interconverted without denaturation. The spectral properties of the beta B2 mutants, as well as their stabilities against denaturants, resemble those of wild-type beta B2-crystallin, thus indicating that the overall peptide fold of the subunits is not changed significantly. We conclude that the peptide linker in beta B2-crystallin is necessary for dimerization, whereas the N- and C-terminal arms appear to be involved in preventing the formation of higher homo-oligomers.     

The X-ray structure of a mutant eye lens beta B2-crystallin with truncated sequence extensions.

beta-Crystallins are oligomeric eye lens proteins that are related to monomeric gamma-crystallins by domain swapping: like gamma-crystallins, they are comprised of two similar domains but they differ in having long sequence extensions. beta B2, a major component of beta-crystallin oligomers, self-associates to a homodimer in solution. In two crystal structures of native beta B2, the protein is a 222-symmetric tetramer of eight domains. It has previously been shown that a mutant of rat beta B2-crystallin, in which the bulk of the N- and C-terminal sequence extensions has been deleted, assembles into dimers and tetramers. Here we present the 3.0 A resolution X-ray structure of the tetramer, beta B2 delta NC1. The mutant tetramer has a very similar set of domain interactions to the native structure. However, the structures differ in the relative orientation of the two sets of four domains. The paired N- and C-terminal domain interface, which is at the heart of the dimer structure, is very similar to the native structure. However, the truncation of the C-terminal extension removes an important tryptophan residue, which prevents the extension from acting as a (non-covalent) linker, as it does in native beta B2. There is a knock-on structural effect that removes a contact between extension and covalent linker, and this appears to cause a small twist in the linker that is amplified into a 20 degrees rotation between sets of paired domains.