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Rainer Jaenicke - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of the calcium loaded Spherulin 3a dimer sheds light on the evolution of the eye lens βγ crystallin domain fold
Structure, 2001Co-Authors: Naomi J Clout, Rainer Jaenicke, Michael Kretschmar, C SlingsbyAbstract:Abstract Background: The βγ-crystallins belong to a superfamily of two-domain proteins found in vertebrate eye lenses, with distant relatives occurring in microorganisms. It has been considered that an eukaryotic stress protein, Spherulin 3a, from the slime mold Physarum polycephalum shares a common one-domain ancestor with crystallins, similar to the one-domain 3-D structure determined by NMR. Results: The X-ray structure of Spherulin 3a shows it to be a tight homodimer, which is consistent with ultracentrifugation studies. The (two-motif) domain fold contains a pair of calcium binding sites very similar to those found in a two-domain prokaryotic βγ-crystallin fold family member, Protein S. Domain pairing in the Spherulin 3a dimer is two-fold symmetric, but quite different in character from the pseudo-two-fold pairing of domains in βγ-crystallins. There is no evidence that the Spherulin 3a single domain can fold independently of its partner domain, a feature that may be related to the absence of a tyrosine corner. Conclusion: Although it is accepted that the vertebrate two-domain βγ-crystallins evolved from a common one-domain ancestor, the mycetezoan single-domain Spherulin 3a, with its unique mode of domain pairing, is likely to be an evolutionary offshoot, perhaps from as far back as the one-motif ancestral stage. The Spherulin 3a protomer stability appears to be dependent on domain pairing. Spherulin-like domain sequences that are found within bacterial proteins associated with virulence are likely to bind calcium.
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lens crystallins and their microbial homologs structure stability and function
Critical Reviews in Biochemistry and Molecular Biology, 2001Co-Authors: Rainer Jaenicke, C SlingsbyAbstract:Referee: Franz Schmid, Biochemicshes Laboratorium, Universitaet Bayeuth, D-95440 Bayeuth, Germanyabg-Crystallins are the major protein components in the vertebrate eye lens — a as a molecular chaperone and b and g as structural proteins. Surprisingly, the latter two share some structural characteristics with a number of microbial stress proteins. The common denominator is not only the Greek key topology of their polypeptide chains but also their high intrinsic stability, which, in certain microbial crystallin homologs, is further enhanced by high-affinity Ca2+-binding. Recent studies of natural and mutant vertebrate bg-crystallins as well as Spherulin 3a from Physarum polycephalum and Protein S from Myxococcus xanthus allowed the correlation of structure and stability of crystallins to be elucidated in some detail. From the thermo-dynamic point of view, stability increments come from (1) local interactions involved in the close packing of the cooperative units, (2) the all-b secondary structure of the Gre...
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stability of a homo dimeric ca 2 binding member of the beta gamma crystallin superfamily dsc measurements on Spherulin 3a from physarum polycephalum
Journal of Molecular Biology, 1999Co-Authors: Michael Kretschmar, Rainer JaenickeAbstract:Abstract Spherulin 3a (S3a) from Physarum polycephalum represents the only known single-domain member of the superfamily of βγ eye-lens crystallins. It shares the typical two Greek-key motif and is stabilized by dimerization and Ca2+-binding. The temperature and denaturant-induced unfolding of S3a in the absence and in the presence of Ca2+ were investigated by differential scanning calorimetry and fluorescence spectroscopy. To accomplish reversibility without chemical modification of the protein during thermal denaturation, the only cysteine residue (Cys4) was substituted by serine; apart from that, the protein was destabilized by adding 0.5–1.8 M guanidinium chloride (GdmCl). The Cys4Ser mutant was found to be indistinguishable from natural S3a. The equilibrium unfolding transitions obey the two-state model according to N2 → 2 U, allowing thermodynamic parameters to be determined by linear extrapolation to zero GdmCl concentration. The corresponding transition temperatures TM for the Ca2+-free and Ca2+-loaded protein were found to be 65 and 85 °C, the enthalpy changes ΔHcal, 800 and 1280 kJ/mol(dimer), respectively. The strong dependencies of TM and ΔHcal on the GdmCl concentration allow the molar heat capacity change ΔCp to be determined. As a result, ΔCp = 18 kJ/(K mol(dimer)) was calculated independent of Ca2+. No significant differences were obtained between the free energy ΔG° calculated from ΔHcal and TM, and extrapolated from the stability curves in the presence of different amounts of denaturant. The free energy derived from thermal unfolding was confirmed by the spectral results obtained from GdmCl-induced equilibrium transitions at different temperatures for the Ca2+-free or the Ca2+-loaded protein, respectively. Within the limits of error, the ΔG° values extrapolated from the transitions of chemical denaturation to zero denaturant concentration are identical with the calorimetric results.
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kinetic and thermodynamic stabilization of the βγ crystallin homolog Spherulin 3a from physarum polycephalum by calcium binding
Journal of Molecular Biology, 1999Co-Authors: Michael Kretschmar, Evamaria Mayr, Rainer JaenickeAbstract:Abstract Globular proteins may be stabilized, either intrinsically, at the various levels of the structural hierarchy, or extrinsically, by ligand binding. In the case of the dormant all-β protein Spherulin 3a (S3a) from the slime mold Physarum polycephalum , binding of calcium ions causes extreme kinetic and thermodynamic stabilization. S3a is the only known single-domain member of the two Greek key superfamily of βγ-crystallins sharing the extreme long-term stability of its homologs in vertebrate eye lens. Spectral analysis allows two Ca 2+ -binding sites with K D =9 μM and 200 μM to be distinguished. Unfolding in the absence and in the presence of Ca 2+ gives evidence for extreme kinetic stabilization of the protein: In the absence of Ca 2+ , the half-time of unfolding in 2.5 M guanidinium chloride (GdmCl) equals 8.3 minutes, whereas in the presence of Ca 2+ , even in 7.5 M GdmCl, it exceeds nine hours. To reach the equilibrium of unfolding in the absence and in the presence of Ca 2+ takes one day and eight weeks, respectively. The corresponding Gibbs free energies (based on the two-state model) are 77 and 135 kJ/mol. Saturation of S3a with Ca 2+ leads to an upward shift of the temperature-induced equilibrium transition by ca 20 deg. C. The in situ Ca 2+ concentration in the spherules is sufficient for the complete complexation of S3a in vivo .
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Homo-dimeric Spherulin 3a: a single-domain member of the beta gamma-crystallin superfamily.
Biological chemistry, 1999Co-Authors: Michael Kretschmar, E.-m. Mayr, Rainer JaenickeAbstract:The beta gamma-crystallin superfamily of eye lens proteins comprises a class of structurally related members with a wide variety of different functions. Common features of these proteins are 1. the Greek-key motif of antiparallel beta-sheets, called the crystallin fold, and 2. the high intrinsic long-term stability. Spherulin 3a (S3a), a dormant protein from the spherules of Physarum polycephalum, is the only known single-domain protein within the beta gamma-crystallin family. Based on sequence homology and 'domain swapping', it has been proposed to represent an evolutionary ancestor of present-day eye lens crystallins. Since S3a is highly expressed in spherulating plasmodia of P. polycephalum under a variety of stress conditions, it can be assumed that the protein may serve as a compatible solute in the cytosol of the slime mold. In order to investigate the stability and other physicochemical properties of a single-domain all-beta protein, we isolated natural S3a. For the large-scale purification, the recombinant protein was cloned and expressed in Escherichia coli. The detailed spectral and biochemical analysis proved the recombinant protein to be authentic. In its native form, S3a is dimeric. Due to its exposed cysteine residues (Cys4), in the absence of reducing agents intermolecular disulfide cross-linking leads to the formation of higher oligomers. In order to preserve the native quaternary structure without aggregation artifacts in denaturation/renaturation experiments, the Cys4-->Ser mutant (S3a C4S) was produced. Both the wild-type protein and its mutant are indistinguishable in their physicochemical properties. At pH 3 - 4, both proteins form a stable compact intermediate (A-state). Concentration-dependent thermal and chemical denaturation showed that the equilibrium unfolding of S3a obeys the simple two-state model with no significant occurrence of folding intermediates.
Michael Kretschmar - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of the calcium loaded Spherulin 3a dimer sheds light on the evolution of the eye lens βγ crystallin domain fold
Structure, 2001Co-Authors: Naomi J Clout, Rainer Jaenicke, Michael Kretschmar, C SlingsbyAbstract:Abstract Background: The βγ-crystallins belong to a superfamily of two-domain proteins found in vertebrate eye lenses, with distant relatives occurring in microorganisms. It has been considered that an eukaryotic stress protein, Spherulin 3a, from the slime mold Physarum polycephalum shares a common one-domain ancestor with crystallins, similar to the one-domain 3-D structure determined by NMR. Results: The X-ray structure of Spherulin 3a shows it to be a tight homodimer, which is consistent with ultracentrifugation studies. The (two-motif) domain fold contains a pair of calcium binding sites very similar to those found in a two-domain prokaryotic βγ-crystallin fold family member, Protein S. Domain pairing in the Spherulin 3a dimer is two-fold symmetric, but quite different in character from the pseudo-two-fold pairing of domains in βγ-crystallins. There is no evidence that the Spherulin 3a single domain can fold independently of its partner domain, a feature that may be related to the absence of a tyrosine corner. Conclusion: Although it is accepted that the vertebrate two-domain βγ-crystallins evolved from a common one-domain ancestor, the mycetezoan single-domain Spherulin 3a, with its unique mode of domain pairing, is likely to be an evolutionary offshoot, perhaps from as far back as the one-motif ancestral stage. The Spherulin 3a protomer stability appears to be dependent on domain pairing. Spherulin-like domain sequences that are found within bacterial proteins associated with virulence are likely to bind calcium.
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stability of a homo dimeric ca 2 binding member of the beta gamma crystallin superfamily dsc measurements on Spherulin 3a from physarum polycephalum
Journal of Molecular Biology, 1999Co-Authors: Michael Kretschmar, Rainer JaenickeAbstract:Abstract Spherulin 3a (S3a) from Physarum polycephalum represents the only known single-domain member of the superfamily of βγ eye-lens crystallins. It shares the typical two Greek-key motif and is stabilized by dimerization and Ca2+-binding. The temperature and denaturant-induced unfolding of S3a in the absence and in the presence of Ca2+ were investigated by differential scanning calorimetry and fluorescence spectroscopy. To accomplish reversibility without chemical modification of the protein during thermal denaturation, the only cysteine residue (Cys4) was substituted by serine; apart from that, the protein was destabilized by adding 0.5–1.8 M guanidinium chloride (GdmCl). The Cys4Ser mutant was found to be indistinguishable from natural S3a. The equilibrium unfolding transitions obey the two-state model according to N2 → 2 U, allowing thermodynamic parameters to be determined by linear extrapolation to zero GdmCl concentration. The corresponding transition temperatures TM for the Ca2+-free and Ca2+-loaded protein were found to be 65 and 85 °C, the enthalpy changes ΔHcal, 800 and 1280 kJ/mol(dimer), respectively. The strong dependencies of TM and ΔHcal on the GdmCl concentration allow the molar heat capacity change ΔCp to be determined. As a result, ΔCp = 18 kJ/(K mol(dimer)) was calculated independent of Ca2+. No significant differences were obtained between the free energy ΔG° calculated from ΔHcal and TM, and extrapolated from the stability curves in the presence of different amounts of denaturant. The free energy derived from thermal unfolding was confirmed by the spectral results obtained from GdmCl-induced equilibrium transitions at different temperatures for the Ca2+-free or the Ca2+-loaded protein, respectively. Within the limits of error, the ΔG° values extrapolated from the transitions of chemical denaturation to zero denaturant concentration are identical with the calorimetric results.
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kinetic and thermodynamic stabilization of the βγ crystallin homolog Spherulin 3a from physarum polycephalum by calcium binding
Journal of Molecular Biology, 1999Co-Authors: Michael Kretschmar, Evamaria Mayr, Rainer JaenickeAbstract:Abstract Globular proteins may be stabilized, either intrinsically, at the various levels of the structural hierarchy, or extrinsically, by ligand binding. In the case of the dormant all-β protein Spherulin 3a (S3a) from the slime mold Physarum polycephalum , binding of calcium ions causes extreme kinetic and thermodynamic stabilization. S3a is the only known single-domain member of the two Greek key superfamily of βγ-crystallins sharing the extreme long-term stability of its homologs in vertebrate eye lens. Spectral analysis allows two Ca 2+ -binding sites with K D =9 μM and 200 μM to be distinguished. Unfolding in the absence and in the presence of Ca 2+ gives evidence for extreme kinetic stabilization of the protein: In the absence of Ca 2+ , the half-time of unfolding in 2.5 M guanidinium chloride (GdmCl) equals 8.3 minutes, whereas in the presence of Ca 2+ , even in 7.5 M GdmCl, it exceeds nine hours. To reach the equilibrium of unfolding in the absence and in the presence of Ca 2+ takes one day and eight weeks, respectively. The corresponding Gibbs free energies (based on the two-state model) are 77 and 135 kJ/mol. Saturation of S3a with Ca 2+ leads to an upward shift of the temperature-induced equilibrium transition by ca 20 deg. C. The in situ Ca 2+ concentration in the spherules is sufficient for the complete complexation of S3a in vivo .
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Homo-dimeric Spherulin 3a: a single-domain member of the beta gamma-crystallin superfamily.
Biological chemistry, 1999Co-Authors: Michael Kretschmar, E.-m. Mayr, Rainer JaenickeAbstract:The beta gamma-crystallin superfamily of eye lens proteins comprises a class of structurally related members with a wide variety of different functions. Common features of these proteins are 1. the Greek-key motif of antiparallel beta-sheets, called the crystallin fold, and 2. the high intrinsic long-term stability. Spherulin 3a (S3a), a dormant protein from the spherules of Physarum polycephalum, is the only known single-domain protein within the beta gamma-crystallin family. Based on sequence homology and 'domain swapping', it has been proposed to represent an evolutionary ancestor of present-day eye lens crystallins. Since S3a is highly expressed in spherulating plasmodia of P. polycephalum under a variety of stress conditions, it can be assumed that the protein may serve as a compatible solute in the cytosol of the slime mold. In order to investigate the stability and other physicochemical properties of a single-domain all-beta protein, we isolated natural S3a. For the large-scale purification, the recombinant protein was cloned and expressed in Escherichia coli. The detailed spectral and biochemical analysis proved the recombinant protein to be authentic. In its native form, S3a is dimeric. Due to its exposed cysteine residues (Cys4), in the absence of reducing agents intermolecular disulfide cross-linking leads to the formation of higher oligomers. In order to preserve the native quaternary structure without aggregation artifacts in denaturation/renaturation experiments, the Cys4-->Ser mutant (S3a C4S) was produced. Both the wild-type protein and its mutant are indistinguishable in their physicochemical properties. At pH 3 - 4, both proteins form a stable compact intermediate (A-state). Concentration-dependent thermal and chemical denaturation showed that the equilibrium unfolding of S3a obeys the simple two-state model with no significant occurrence of folding intermediates.
Max Bergoin - One of the best experts on this subject based on the ideXlab platform.
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THE MELOLONTHA-MELOLONTHA ENTOMOPOXVIRUS (MMEPV) FUSOLIN IS RELATED TO THE FUSOLINS OF LEPIDOPTERAN EPVS AND TO THE 37K BACULOVIRUS GLYCOPROTEIN
Virology, 1995Co-Authors: Laurent Gauthier, Jean Claude Veyrunes, Francois Cousserans, Max BergoinAbstract:Abstract We have cloned and sequenced a 1.7-kbp DNA fragment of the MmEPV genome encompassing the major polypeptide of the spindle-shaped inclusions gene termed fusolin. The sequence contained a single open reading frame of 1203 nt capable of coding for a polypeptide of 45.8 kDa. The 13 N-terminal amino acid (aa) residues were hydrophobic and could act as a signal peptide. The aa sequence also contained 13 cysteine residues very likely involved in paracrystal formation. This sequence showed significant homologies with the fusolins of two lepidopteran EPVs, the Choristoneura biennis EPV (CbEPV) and the Heliothis armigera EPV, and also with the 37K glycoproteins of Autographa californica and Orgyia pseudotsugata baculoviruses. No homology was found between the MmEPV fusolin and the 100K MmEPV Spherulin, nor with the 11OK polypeptide of the CbEPV and Amsacta moorei EPV spheroidins. These data were confirmed by Western blot analysis. Transfection of vaccinia-infected mammalian cells with a plasmid encompassing the fusolin sequence plus the upstream regulatory region resulted in transient expression of the gene. This indicated that the vaccinia transcription machinery is able to transcribe the fusolin gene. The fusolin was also expressed in insect cells via a recombinant baculovirus.
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cloning and sequencing of the Spherulin gene the occlusion body major polypeptide of the melolontha melolontha entomopoxvirus mmepv
Virology, 1994Co-Authors: Patrick Sanz, Jean Claude Veyrunes, Francois Cousserans, Max BergoinAbstract:Abstract In the late stage of infection, virions of the Melolontha melolontha entomopoxvirus ( Mm EPV) are occluded into cytoplasmic paracrystalline proteinaceous occlusion bodies designated spherules (A. Amargier, C. Vago, G. Meynadier, 1964, Mikroskopie 19, 309-315). We have cloned and sequenced a 4-kpb DNA fragment of the Mm EPV genome encompassing the spherule major protein gene named Spherulin. The Spherulin gene contains an open reading frame able to code for a 942-amino-acid (aa) polypeptide (MW 109 kDa), consistent with a size above 100 kDa determined by SDS-PAGE for purified Spherulin. The Mm EPV Spherulin showed more than 40% aa homology with the Amsacta moorei EPV ( Am EPV) spheroidin and shared homologies with the partially sequenced Choristoneura biennis EPV ( Cb EPV) spheroidin, indicating that this biologically important polypeptide is well conserved among EPVs infecting phylogenetically as distant groups of insects as lepidoptera and coleoptera. Western blot analyses confirmed the relationships between the three polypeptides. in contrast, no homology was detected between the Mm EPV Spherulin and EPV fusolins or vertebrate poxvirus A-type inclusion proteins. The 45 bases upstream from the ATG initiation codon of Spherulin shared 60% homology with the vaccinia virus late promoters including the highly conserved TAAATG consensus sequence. Furthermore, the 5′ extremity of the Spherulin mRNA consisted of a poly(A) tract of about 20 nucleotides just upstream from the AUG translational initiation codon. These are characteristic features of vertebrate poxvirus late mRNAs suggesting similar modalities of gene expression for vertebrate and insect poxvirus genomes.
C Slingsby - One of the best experts on this subject based on the ideXlab platform.
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crystal structure of the calcium loaded Spherulin 3a dimer sheds light on the evolution of the eye lens βγ crystallin domain fold
Structure, 2001Co-Authors: Naomi J Clout, Rainer Jaenicke, Michael Kretschmar, C SlingsbyAbstract:Abstract Background: The βγ-crystallins belong to a superfamily of two-domain proteins found in vertebrate eye lenses, with distant relatives occurring in microorganisms. It has been considered that an eukaryotic stress protein, Spherulin 3a, from the slime mold Physarum polycephalum shares a common one-domain ancestor with crystallins, similar to the one-domain 3-D structure determined by NMR. Results: The X-ray structure of Spherulin 3a shows it to be a tight homodimer, which is consistent with ultracentrifugation studies. The (two-motif) domain fold contains a pair of calcium binding sites very similar to those found in a two-domain prokaryotic βγ-crystallin fold family member, Protein S. Domain pairing in the Spherulin 3a dimer is two-fold symmetric, but quite different in character from the pseudo-two-fold pairing of domains in βγ-crystallins. There is no evidence that the Spherulin 3a single domain can fold independently of its partner domain, a feature that may be related to the absence of a tyrosine corner. Conclusion: Although it is accepted that the vertebrate two-domain βγ-crystallins evolved from a common one-domain ancestor, the mycetezoan single-domain Spherulin 3a, with its unique mode of domain pairing, is likely to be an evolutionary offshoot, perhaps from as far back as the one-motif ancestral stage. The Spherulin 3a protomer stability appears to be dependent on domain pairing. Spherulin-like domain sequences that are found within bacterial proteins associated with virulence are likely to bind calcium.
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lens crystallins and their microbial homologs structure stability and function
Critical Reviews in Biochemistry and Molecular Biology, 2001Co-Authors: Rainer Jaenicke, C SlingsbyAbstract:Referee: Franz Schmid, Biochemicshes Laboratorium, Universitaet Bayeuth, D-95440 Bayeuth, Germanyabg-Crystallins are the major protein components in the vertebrate eye lens — a as a molecular chaperone and b and g as structural proteins. Surprisingly, the latter two share some structural characteristics with a number of microbial stress proteins. The common denominator is not only the Greek key topology of their polypeptide chains but also their high intrinsic stability, which, in certain microbial crystallin homologs, is further enhanced by high-affinity Ca2+-binding. Recent studies of natural and mutant vertebrate bg-crystallins as well as Spherulin 3a from Physarum polycephalum and Protein S from Myxococcus xanthus allowed the correlation of structure and stability of crystallins to be elucidated in some detail. From the thermo-dynamic point of view, stability increments come from (1) local interactions involved in the close packing of the cooperative units, (2) the all-b secondary structure of the Gre...
Yogendra Sharma - One of the best experts on this subject based on the ideXlab platform.
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three dimensional domain swapping in nitrollin a single domain βγ crystallin from nitrosospira multiformis controls protein conformation and stability but not dimerization
Journal of Molecular Biology, 2009Co-Authors: Penmatsa Aravind, Yogendra Sharma, Shashi Kumar Suman, Amita Mishra, Rajan SankaranarayananAbstract:The βγ-crystallin superfamily has a well-characterized protein fold, with several members found in both prokaryotic and eukaryotic worlds. A majority of them contain two βγ-crystallin domains. A few examples, such as ciona crystallin and Spherulin 3a exist that represent the eukaryotic single-domain proteins of this superfamily. This study reports the high-resolution crystal structure of a single-domain βγ-crystallin protein, nitrollin, from the ammonium-oxidizing soil bacterium Nitrosospira multiformis. The structure retains the characteristic βγ-crystallin fold despite a very low sequence identity. The protein exhibits a unique case of homodimerization in βγ-crystallins by employing its N-terminal extension to undergo three-dimensional (3D) domain swapping with its partner. Removal of the swapped strand results in partial loss of structure and stability but not dimerization per se as determined using gel filtration and equilibrium unfolding studies. Overall, nitrollin represents a distinct single-domain prokaryotic member that has evolved a specialized mode of dimerization hitherto unknown in the realm of βγ-crystallins.
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Calcium-binding to lens βB2- and βA3-crystallins suggests that all β-crystallins are calcium-binding proteins
FEBS Journal, 2007Co-Authors: Maroor K. Jobby, Yogendra SharmaAbstract:Crystallins are the major proteins of a mammalian eye lens. The topologically similar eye lens proteins, β- and γ-crystallins, are the prototype and founding members of the βγ-crystallin superfamily. βγ-Crystallins have until recently been regarded as structural proteins. However, the calcium-binding properties of a few members and the potential role of βγ-crystallins in fertility are being investigated. Because the calcium-binding elements of other member proteins, such as Spherulin 3a, are not present in βB2-crystallin and other βγ-crystallins from fish and mammalian genomes, it was argued that lens βγ-crystallins should not bind calcium. In order to probe whether β-crystallins can bind calcium, we selected one basic (βB2) and one acidic (βA3) β-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 βB2- and βA3-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 βB2- and βA3-crystallins, we suggest that all β-crystallins bind calcium. Our results have important implications for understanding the calcium-related cataractogenesis and maintenance of ionic homeostasis in the lens.
