Heteromeric formation with ßA3 protects the low thermal stability of ßB1-L116P.

BACKGROUND/AIMS: Congenital cataract is the leading cause of visual disability and blindness in childhood. ßB1-crystallin (CRYBB1) comprises about 1/10th of crystallin structural proteins, forming heteromers to maintain lens transparency. We previously reported a CRYBB1 mutation (c.347T>C, p.L116P) affecting 16 patients in a congenital nuclear cataract family. In this study, we investigate the underlying pathogenic mechanism of ßB1-L116P. METHODS: Protein isolation, size-exclusion chromatography, spectroscopy, Uncle stability screens and molecular dynamics simulations were used to assess ßA3- and ßB1-crystallin thermal stability, structural properties and heteromer formation. RESULTS: Cells that overexpressed ßB1-L116P tended to form aggregates and precipitations under heat-shock stress. Thermal denaturation and time-dependent turbidity experiments showed that thermal stability was significantly impaired. Moreover, protein instability appeared to increase with elevated concentrations detected by the Uncle system. Additionally, ßA3 had a relative protective effect on ßB1-L116P after heteromers were formed, although ßA3 was relatively unstable and was usually protected by basic ß-crystallins. Molecular dynamic simulations revealed that L116P mutation altered the hydrophobic residues at the surface around the mutant site, providing solvents more access to the internal and hydrophobic parts of the protein. CONCLUSIONS: Decreased ßB1-crystallin thermal stability in the presence of the cataract-related L116P mutation contributes significantly to congenital cataract formation. Moreover, its formation of heteromers with ßA3 protects against the low thermal stability of ßB1-L116P.

Congenital cataract-causing mutation ßB1-L116P is prone to amyloid fibrils aggregation and protease degradation with low structural stability.

Congenital cataract, a common disease with lens opacification, causes blindness in the newborn worldwide and is mainly caused by abnormal aggregation of crystallin. As the main structural protein in the mammalian lens, ßB1-crystallin has an important role in the maintenance of lens transparency. Recently, the L116P mutation in ßB1-CRY was found in a Chinese family with congenital nuclear cataracts, while its underlying pathogenic mechanism remains unclear. In the current study, the ßB1 wild-type protein was purified, and the mutated form, ßB1-L116P, was examined for examining the effect on structural stability and susceptibility against environmental stresses. Our results reveal low solubility and structural stability of ßB1-L116P at physiological temperature, which markedly impaired the protein structure and the oligomerization of ßB1-crystallin. Under guanidine hydrochloride-induced denaturing conditions, ßB1-L116P mutation perturbed the protein unfolding process, making it prone to amyloid fibrils aggregation. More importantly, the L116P mutation increased susceptibility of ßB1-crystallin against UV radiation. ßB1-L116P overexpression led to the formation of more serious intracellular aggresomes under UV radiation or oxidative stress. Furthermore, the ßB1-L116P mutation increased the sensitivity to the proteolysis process. These results indicate that the low structural stability, susceptibility to amyloid fibrils aggregation, and protease degradation of ßB1-L116P may contribute to cataract development and associated symptoms.

ßB2 W151R mutant is prone to degradation, aggregation and exposes the hydrophobic side chains in the fourth Greek Key motif.

Studies have established that congenital cataract is the major cause of blindness in children across the globe. The ß-crystallin protein family is the richest and most soluble structural protein in the lens. Their solubility and stability are essential in maintaining lens transparency. In this study, we identified a novel ßB2 mutation W151R in a rare progressive cortical congenital cataract family and explored its pathogenesis using purified protein and mutant related cataract-cell models. Due to its low solubility and poor structural stability, the ßB2 W151R mutation was prone to aggregation. Moreover, the W151R mutation enhanced the exposure of the hydrophobic side chains in the fourth Greek Key motif, which were readily degraded by trypsin. However, upon the administration of lanosterol, the negative effect of the W151R mutation was reversed. Therefore, lanosterol is a potential therapeutic option for cataracts.

Exploring the folding process of human ßB2-crystallin using multiscale molecular dynamics and the Markov state model.

Adequate knowledge of protein conformations is crucial for understanding their function and their association properties with other proteins. The cataract disease is correlated with conformational changes in key proteins called crystallins. These changes are due to mutations or post-translational modifications that may lead to protein unfolding, and thus the formation of aggregate states. Human ßB2-crystallin (HßB2C) is found in high proportion in the eye lens, and its mutations are related to some cataracts. HßB2C also associates into dimers, tetramers, and other higher-order supramolecular complexes. However, it is the only protein of the ßγ-crystallin family that has been found in an extended conformation. Therefore, we hypothesize that the extended conformation is not energetically favourable and that HßB2C may adopt a closed (completely folded) conformation, similar to the other members of the ßγ-crystallin family. To corroborate this hypothesis, we performed extensive molecular dynamics simulations of HßB2C in its monomeric and dimeric conformations, using all-atom and coarse-grained scales. We employed Markov state model (MSM) analysis to characterize the conformational and kinetically relevant states in the folding process of monomeric HßB2C. The MSM analysis clearly shows that HßB2C adopts a completely folded structure, and this conformation is the most kinetically and energetically favourable one. In contrast, the extended conformations are kinetically unstable and energetically unfavourable. Our MSM analysis also reveals a key metastable state, which is particularly interesting because it is from this state that the folded state is reached. The folded state is stabilized by the formation of two salt bridges between the residue-pairs E74-R187 and R97-E166 and the two hydrophobic residue-pairs V59-L164 and V72-V151. Furthermore, free energy surface (FES) analysis revealed that the HßB2C dimer with both monomers in a closed conformation (face-en-face dimer) is energetically more stable than the domain-swapped dimer (crystallographic structure). The results presented in this report shed light on the molecular details of the folding mechanism of HßB2C in an aqueous environment and may contribute to interpreting different experimental findings. Finally, a detailed knowledge of HßB2C folding may be key to the rational design of potential molecules to treat cataract disease.

Predicted aggregation-prone region (APR) in ßB1-crystallin forms the amyloid-like structure and induces aggregation of soluble proteins isolated from human cataractous eye lens.

The aggregation of ß-crystallins in the human eye lens constitutes a critical step during the development of cataract. We anticipated that the presence of Aggregation-Prone Regions (APRs) in their primary structure, which might be responsible for conformational change required for the self-assembly. To examine the presence of APRs, we systematically analyzed the primary structures of ß-crystallins. Out of seven subtypes, the ßB1-crystallin found to possess the highest aggregation score with 9 APRs in its primary structure. To confirm the amyloidogenic nature of these newly identified APRs, we further studied the aggregation behavior of one of the APRs spanning from 174 to 180 residues (174LWVYGFS180) of ßB1-crystallin, which is referred as ßB1(174-180). Under in vitro conditions, the synthetic analogue of ßB1(174-180) peptide formed visible aggregates and displayed high Congo red (CR) bathochromic shift, Thioflavin T (ThT) binding and fibrilar morphology under transmission electron microscopy, which are the typical characteristics of amyloids. Further, the aggregated ßB1(174-180) was found to induce aggregation of the soluble fraction of proteins isolated from the human cataractous lens. This observation suggests that the presence of APRs in ßB1-crystallin might be serving as one of the intrinsic supplementary factors responsible for constitutive aggregation behavior of ßB1-crystallin and development of cataract.

Crybb2 Mutations Consistently Affect Schizophrenia Endophenotypes in Mice.

As part of the ßγ-superfamily, ßB2-crystallin (CRYBB2) is an ocular structural protein in the lens, and mutation of the corresponding gene can cause cataracts. CRYBB2 also is expressed in non-lens tissue such as the adult mouse brain and is associated with neuropsychiatric disorders such as schizophrenia. Nevertheless, the robustness of this association as well as how CRYBB2 may contribute to disease-relevant phenotypes is unknown. To add further clarity to this issue, we performed a comprehensive analysis of behavioral and neurohistological alterations in mice with an allelic series of mutations in the C-terminal end of the Crybb2 gene. Behavioral phenotyping of these three ßB2-mutant lines Crybb2O377, Crybb2Philly, and Crybb2Aey2 included assessment of exploratory activity and anxiety-related behavior in the open field, sensorimotor gating measured by prepulse inhibition (PPI) of the acoustic startle reflex, cognitive performance measured by social discrimination, and spontaneous alternation in the Y-maze. In each mutant line, we also quantified the number of parvalbumin-positive (PV+) GABAergic interneurons in selected brain regions that express CRYBB2. While there were allele-specific differences in individual behaviors and affected brain areas, all three mutant lines exhibited consistent alterations in PPI that paralleled alterations in the PV+ cell number in the thalamic reticular nucleus (TRN). The direction of the PPI change mirrored that of the TRN PV+ cell number thereby suggesting a role for TRN PV+ cell number in modulating PPI. Moreover, as both altered PPI and PV+ cell number are schizophrenia-associated endophenotypes, our result implicates mutated Crybb2 in the development of this neuropsychiatric disorder.

Introduction of an extra tryptophan fluorophore by cataract-associating mutations destabilizes ßB2-crystallin and promotes aggregation.

ß/γ-Crystallins are predominant structural proteins in vertebrate lens with unique properties of extremely high solubility, long-term stability and resistance to UV damage. Four conserved Trp residues in ß/γ-crystallins account for UV absorbance and thereafter fluorescence quenching to avoid photodamage. Herein we found that ßB2-crystallin Trp fluorescence was greatly enhanced by the introduction of an extra unquenched Trp fluorophore by cataract-associated mutations S31W and R145W. Both mutations impaired oligomerization, decreased stability and promote thermal aggregation, while S31W was more deleterious. S31W accelerated ßB2-crystallin aggregation under UV damaging conditions, whereas R145W delayed. These observations suggested that the introduction of an extra Trp fluorophore had complicated effects on ßB2-crystallin stability and aggregation against various stresses. Our findings highlight that the number of Trp fluorophores in ß/γ-crystallin is evolutionarily optimized to exquisitely perform their structural roles in the lens.

Identification of Isomeric Aspartate residues in ßB2-crystallin from Aged Human Lens.

Many post-translational modifications such as oxidation, deamidation and isomerization of amino acid residues occur in lens proteins with aging. One such modification, isomerization of aspartate in lens α-crystallin, has been well studied by amino acid enantiomer analysis and LC-MS/MS. LC-MS/MS can quickly and easily identify D- and L-amino acid-containing peptides without purification of lens protein mixtures. However, this method has a weak point in that isomeric peptides of major components are detected predominantly, while those from minor proteins such as ß- and γ-crystallins have not been fully determined. Therefore, the isomerization of amino acid residues in ß- and γ-crystallin families has been little studied. To solve those problems and detect the isomerization of Asp residues in lens ßB2-crystallin, the main component of the ß-crystallin family, here we have developed steps for sample fractionation before d/l analysis based on either LC-MS/MS or amino acid derivatization to diastereoisomers followed by RP-HPLC. To capture a small amount of peptide, a multiple reaction monitoring (MRM) method based on quadrupole MS/MS (Q-MS) was applied to the water-soluble fraction of whole lens. The d/l analysis based on both LC-MS/MS and diastereoisomer formation showed the presence of multiple isomerization sites, including Asp4, Asp83, Asp92 and Asp192, in ßB2-crystallin in aged lens. These isomerization sites were confirmed to exist in an age-dependent manner by Q-MS. Synthetic peptides of ßB2-crystallin containing different isomers of Asp showed differential elution profiles during RP-HPLC, indicating differences in the local structure or hydrophobicity of Asp-isomer-containing peptides. These results suggest that the isomerization sites are distributed on exposed regions of ßB2-crystallin and thus likely to have an impact on crystallin subunit-subunit interactions, induce abnormal crystallin aggregation, and contribute to senile cataract formation in aged lens.

Peptides for targeting ßB2-crystallin fibrils.

Crystallins are a major family of proteins located within the lens of the eye. Cataracts are thought to be due to the formation of insoluble fibrillar aggregates, which are largely composed of proteins from the crystallin family. Today the only cataract treatment that exists is surgery and this can be difficult to access for individuals in the developing world. Development of novel pharmacotherapeutic approaches for the treatment of cataract rests on the specific targeting of these structures. ßB2-crystallin, a member of ß-crystallin family, is a large component of the crystallin proteins within the lens, and as such was used to form model fibrils in vitro. Peptides were identified, using phage display techniques, that bound to these fibrils with high affinity. Fibrillation of recombinantly expressed human ßB2-crystallin was performed in 10% (v/v) trifluoroethanol (TFE) solution (pH 2.0) at various temperatures, and its amyloid-like structure was confirmed using Thioflavin-T (ThT) assay, transmission electron microscopy (TEM), and X-ray fiber diffraction (XRFD) analysis. Affinity of identified phage-displayed peptides were analyzed using enzyme-linked immunosorbent assay (ELISA). Specific binding of a cyclic peptide (CKQFKDTTC) showed the highest affinity, which was confirmed using a competitive inhibition assay.

Effects of cataract-causing mutations W59C and W151C on ßB2-crystallin structure, stability and folding.

ß/γ-Crystallins, the predominant structural proteins in vertebrate lens with lifelong stability to maintain lens transparency, share a high similarity in their primary sequences and tertiary structures. Four conserved Trp residues have been shown to be important to γ-crystallin structure, stability and protection against UV irradiation, whereas their roles in ß-crystallins remain elusive. Herein we found that two congenital cataract-causing mutations, W59C and W151C, dramatically decreased ßB2-crystallin solubility and stability against thermal and guanidine hydrochloride-induced denaturation. The two mutated proteins were prone to form aggregates when irradiated by UV light in the tubes or exogenously expressed in the cells. Although W59 and W151 are structurally identical in ß/γ-crystallin domains, substituting them by Cys led to dissimilar influences on ßB2-crystallin stability. Our results suggested that the conserved Trp residues might play a more crucial role in the correct folding and structural integrity of ß-crystallin domains than in γ-crystallins.

Human ßB2-Crystallin Forms a Face-en-Face Dimer in Solution: An Integrated NMR and SAXS Study.

ßγ-Crystallins are long-lived eye lens proteins that are crucial for lens transparency and refractive power. Each ßγ-crystallin comprises two homologous domains, which are connected by a short linker. γ-Crystallins are monomeric, while ß-crystallins crystallize as dimers and multimers. In the crystal, human ßB2-crystallin is a domain-swapped dimer while the N-terminally truncated ßB1-crystallin forms a face-en-face dimer. Combining and integrating data from multi-angle light scattering, nuclear magnetic resonance, and small-angle X-ray scattering of full-length and terminally truncated human ßB2-crystallin in solution, we show that both these ßB2-crystallin proteins are dimeric, possess C2 symmetry, and are more compact than domain-swapped dimers. Importantly, no inter-molecular paramagnetic relaxation enhancement effects compatible with domain swapping were detected. Our collective experimental results unambiguously demonstrate that, in solution, human ßB2-crystallin is not domain swapped and exhibits a face-en-face dimer structure similar to the crystal structure of truncated ßB1-crystallin.

Cataract-causing mutation S228P promotes ßB1-crystallin aggregation and degradation by separating two interacting loops in C-terminal domain.

ß/γ-Crystallins are predominant structural proteins in the cytoplasm of lens fiber cells and share a similar fold composing of four Greek-key motifs divided into two domains. Numerous cataract-causing mutations have been identified in various ß/γ-crystallins, but the mechanisms underlying cataract caused by most mutations remains uncharacterized. The S228P mutation in ßB1-crystallin has been linked to autosomal dominant congenital nuclear cataract. Here we found that the S228P mutant was prone to aggregate and degrade in both of the human and E. coli cells. The intracellular S228P aggregates could be redissolved by lanosterol. The S228P mutation modified the refolding pathway of ßB1-crystallin by affecting the formation of the dimeric intermediate but not the monomeric intermediate. Compared with native ßB1-crystallin, the refolded S228P protein had less packed structures, unquenched Trp fluorophores and increased hydrophobic exposure. The refolded S228P protein was prone to aggregate at the physiological temperature and decreased the protective effect of ßB1-crystallin on ßA3-crystallin. Molecular dynamic simulation studies indicated that the mutation decreased the subunit binding energy and modified the distribution of surface electrostatic potentials. More importantly, the mutation separated two interacting loops in the C-terminal domain, which shielded the hydrophobic core from solvent in native ßB1-crystallin. These two interacting loops are highly conserved in both of the N- and C-terminal domains of all ß/γ-crystallins. We propose that these two interacting loops play an important role in the folding and structural stability of ß/γ-crystallin domains by protecting the hydrophobic core from solvent access.

Congenital microcornea-cataract syndrome-causing mutation X253R increases ßB1-crystallin hydrophobicity to promote aggregate formation.

The high solubility and lifelong stability of crystallins are crucial to the maintenance of lens transparency and optical properties. Numerous crystallin mutations have been linked to congenital cataract, which is one of the leading causes of newborn blindness. Besides cataract, several crystallin mutations have also been linked to syndromes such as congenital microcornea-cataract syndrome (CMCC). However, the molecular mechanism of CMCC caused by crystallin mutations remains elusive. In the present study, we investigated the mechanism of CMCC caused by the X253R mutation in ßB1-crystallin. The exogenously expressed X253R proteins were prone to form p62-negative aggregates in HeLa cells, strongly inhibited cell proliferation and induced cell apoptosis. The intracellular X253R aggregates could be successfully redissolved by lanosterol but not cholesterol. The extra 26 residues at the C-terminus of ßB1-crystallin introduced by the X253R mutation had little impact on ßB1-crystallin structure and stability, but increased ßB1-crystallin hydrophobicity and decreased its solubility. Interestingly, the X253R mutant fully abolished the aggregatory propensity of ßB1- and ßA3/ßB1-crystallins at high temperatures, suggesting that X253R was an aggregation-inhibition mutation of ß-crystallin homomers and heteromers in dilute solutions. Our results suggest that an increase in hydrophobicity and a decrease in solubility might be responsible for cataractogenesis induced by the X253R mutation, while the cytotoxic effect of X253R aggregates might contribute to the defects in ocular development. Our results also highlight that, at least in some cases, the aggregatory propensity in dilute solutions could not fully mimic the behaviours of mutated proteins in the crowded cytoplasm of the cells.

Stability of the ßB2B3 crystallin heterodimer to increased oxidation by radical probe and ion mobility mass spectrometry.

Ion mobility mass spectrometry was employed to study the structure of the ßB2B3-crystallin heterodimer following oxidation through its increased exposure to hydroxyl radicals. The results demonstrate that the heterodimer can withstand limited oxidation through the incorporation of up to some 10 oxygen atoms per subunit protein without any appreciable change to its average collision cross section and thus conformation. These results are in accord with the oxidation levels and timescales applicable to radical probe mass spectrometry (RP-MS) based protein footprinting experiments. Following prolonged exposure, the heterodimer is increasingly degraded through cleavage of the backbone of the subunit crystallins rather than denaturation such that heterodimeric structures with altered conformations and ion mobilities were not detected. However, evidence from measurements of oxidation levels within peptide segments, suggest the presence of some aggregated structure involving C-terminal domain segments of ßB3 crystallin across residues 115-126 and 152-166. The results demonstrate, for the first time, the ability of ion mobility in conjunction with RP-MS to investigate the stability of protein complexes to, and the onset of, free radical based oxidative damage that has important implications in cataractogenesis.

Cataract-linked mutation R188H promotes ßB2-crystallin aggregation and fibrillization during acid denaturation.

Cataract is characterized by the formation of light-scattering protein aggregates in the lens. ß/γ-Crystallins are the predominant structural proteins in the cytosol of lens fiber cells, and more than fifty ß/γ-crystallin mutations have been linked to autosomal dominant congenital cataract. However, the structural role of these mutations in the formation of the core structures of amorphous aggregates or amyloid-like fibrils has not been elucidated yet. In this research, we studied the effects of the V187M and R188H mutations on the aggregation and fibrillization of ßB2-crystallin during acid denaturation. The behavior of V187M was the same as the WT protein, suggesting that the residue at position 187 contributed little to the aggregation/fibrillization process. R188H promoted the formation of amorphous aggregates at pH above 3 and accelerated fibrillization at pH 3. The distinct behaviors of the mutants suggested that the residue at position 188 might play a regulatory role in ßB2-crystallin aggregation/fibrillization but not reside in the core of the aggregates/fibrils.

The importance of the last strand at the C-terminus in ßB2-crystallin stability and assembly.

Congenital cataract is the leading cause of childhood blindness worldwide. Investigations of the effects of inherited mutations on protein structure and function not only help us to understand the molecular mechanisms underlying congenital hereditary cataract, but also facilitate the study of complicated cataract and non-lens abnormities caused by lens-specific genes. In this research, we studied the effects of the V187M, V187E and R188H mutations on ßB2-crystallin structure and stability using a combination of biophysical, cellular and molecular dynamic simulation analysis. Both V187 and R188 are located at the last strand of ßB2-crystallin Greek-key motif 4. All of the three mutations promoted ßB2-crystallin aggregation in vitro and at the cellular level. These three mutations affected ßB2-crystallin quite differentially: V187M influenced the hydrophobic core of the C-terminal domain, V187E was a Greek-key motif breaker with the disruption of the backbone H-bonding network, while R188H perturbed the dynamic oligomeric equilibrium by dissociating the dimer and stabilizing the tetramer. Our results highlighted the importance of the last strand in the structural integrity, folding, assembly and stability of ß-crystallins. More importantly, we proposed that the perturbation of the dynamic equilibrium between ß-crystallin oligomers was an important mechanism of congenital hereditary cataract. The selective stabilization of one specific high-order oligomer by mutations might also be deleterious to the stability and folding of the ß-crystalllin homomers and heteromers. The long-term structural stability and functional maintenance of ß-crystallins are achieved by the precisely regulated oligomeric equilibrium.

A missense mutation in CRYBB2 leads to progressive congenital membranous cataract by impacting the solubility and function of ßB2-crystallin.

Congenital cataract is a major cause of visual impairment and childhood blindness. The solubility and stability of crystallin proteins play critical roles in maintaining the optical transparency of the lens during the life span. Previous studies have shown that approximately 8.3%~25% of congenital cataracts are inherited, and mutations in crystallins are the most common. In this study, we attempted to identify the genetic defect in a four-generation family affected with congenital cataracts. The congenital cataract phenotype of this four-generation family was identified as membranous cataract by slit-lamp photography. Mutation screening of the candidate genes detected a heterozygous c.465G → C change in the exon6 of the ßB2-crystallin gene (CRYBB2) in all family members affected with cataracts, resulting in the substitution of a highly conserved Tryptophan to Cystine (p.W151C). The mutation was confirmed by restriction fragment length polymorphism (RFLP) analysis and found that the transition resulted in the absence of a BslI restriction site in the affected members of the pedigree. The outcome of PolyPhen-2 and SIFT analysis predicted that this W151C mutation would probably damage to the structure and function of ßB2-crystallin. Wild type (wt) and W151C mutant ßB2-crystallin were expressed in human lens epithelial cells (HLECs), and the fluorescence results showed that Wt-ßB2-crystallin was evenly distributed throughout the cells, whereas approximately 34.7% of cells transfected with the W151C mutant ßB2-crystallin formed intracellular aggregates. Taken together, these data suggest that the missense mutation in CRYBB2 gene leads to progressive congenital membranous cataract by impacting the solubility and function of ßB2-crystallin.

Changes in solvent accessibility of wild-type and deamidated ßB2-crystallin following complex formation with αA-crystallin.

Aberrant protein interactions can lead to aggregation and insolubilization, such as occurs during cataract formation. Deamidation, a prevalent age-related modification in the lens of the eye, decreases stability of the major lens proteins, crystallins. The mechanism of deamidation altering interactions between αA-crystallin and ßB2-crystallin was investigated by detecting changes in solvent accessibility upon complex formation during heating. Solvent accessibility was determined by measuring hydrogen/deuterium exchange levels of backbone amides by high-resolution mass spectrometry. Deuterium levels in wild type ßB2-crystallin increased 50-60% in both domains following complex formation with αA-crystallin. This increased solvent accessibility indicated a general loosening along the backbone amides. Peptides with the greatest deuterium increases were located at the buried monomer-monomer interface, suggesting that the ßB2 dimer was disrupted. The only region where the deuterium levels decreased was in ßB2 peptide 123-139, containing an outside loop, and may be a potential site of interaction with αA. Mimicking deamidation at the ßB2 dimer interface prevented complex formation with αA. When temperatures were lowered, an αA/ßB2 Q70E/Q162E complex formed with similar solvent accessibilities as αA/WT ßB2. Deamidation did not disrupt specific αA/ßB2 interactions but favored aggregation before complex formation with αA. We conclude that deamidation contributes to cataract formation through destabilization of crystallins before they can be rescued by α-crystallin.

ßB1-crystallin: thermodynamic profiles of molecular interactions.

BACKGROUND: ß-Crystallins are structural proteins maintaining eye lens transparency and opacification. Previous work demonstrated that dimerization of both ßA3 and ßB2 crystallins (ßA3 and ßB2) involves endothermic enthalpy of association (∼8 kcal/mol) mediated by hydrophobic interactions. METHODOLOGY/PRINCIPAL FINDINGS: Thermodynamic profiles of the associations of dimeric ßA3 and ßB1 and tetrameric ßB1/ßA3 were measured using sedimentation equilibrium. The homo- and heteromolecular associations of ßB1 crystallin are dominated by exothermic enthalpy (-13.3 and -24.5 kcal/mol, respectively). CONCLUSIONS/SIGNIFICANCE: Global thermodynamics of ßB1 interactions suggest a role in the formation of stable protein complexes in the lens via specific van der Waals contacts, hydrogen bonds and salt bridges whereas those ß-crystallins which associate by predominately hydrophobic forces participate in a weaker protein associations.

The benefits of being ß-crystallin heteromers: ßB1-crystallin protects ßA3-crystallin against aggregation during co-refolding.

ß-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 ß-crystallins, ßA3-crystallin and ßB1-crystallin, an acidic and a basic ß-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 ß-crystallin facilitates the folding of acidic ß-crystallin. Equilibrium folding studies revealed that the ßA3-crystallin and ßB1-crystallin homomers and the ßA3/ßB1-crystallin heteromer all undergo similar five-state folding pathways which include one dimeric and two monomeric intermediates. ßA3-Crystallin was found to be the most unstable among the three proteins, and the transition curve of ßA3/ßB1-crystallin was close to that of ßB1-crystallin. The dimeric intermediate may be a critical determinant in the aggregation process and thus is crucial to the lifelong stability of the ß-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, ßA3-crystallin, the only protein whose refolding is challenged by serious aggregation, can be protected by ßB1-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 ßB1-crystallin. These findings suggested that the formation of ß-crystallin heteromers not only stabilizes the unstable acidic ß-crystallin but also protects them against aggregation during refolding from the stress-denatured states.