Primary structure of beta s-crystallin from human lens.
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ABSTRACT:
SUBMITTER: Zarina S
PROVIDER: S-EPMC1133175 | biostudies-other | 1992 Oct
REPOSITORIES: biostudies-other
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Project description:According to the World Health Organization, cataracts account for half of the blindness in the world, with the majority occurring in developing countries. A cataract is a clouding of the lens of the eye due to light scattering of precipitated lens proteins or aberrant cellular debris. The major proteins in the lens are crystallins, and they are extensively deamidated during aging and cataracts. Deamidation has been detected at the domain and monomer interfaces of several crystallins during aging. The purpose of this study was to determine the effects of two potential deamidation sites at the predicted interface of the betaA3-crystallin dimer on its structure and stability. The glutamine residues at the reported in vivo deamidation sites of Q180 in the C-terminal domain and at the homologous site Q85 in the N-terminal domain were substituted with glutamic acid residues by site-directed mutagenesis. Far-UV and near-UV circular dichroism spectroscopy indicated that there were subtle differences in the secondary structure and more notable differences in the tertiary structure of the mutant proteins compared to that of the wild type betaA3-crystallin. The Q85E/Q180E mutant also was more susceptible to enzymatic digestion, suggesting increased solvent accessibility. These structural changes in the deamidated mutants led to decreased stability during unfolding in urea and increased precipitation during heat denaturation. When simulating deamidation at both residues, there was a further decrease in stability and loss of cooperativity. However, multiangle-light scattering and quasi-elastic light scattering experiments showed that dimer formation was not disrupted, nor did higher-order oligomers form. These results suggest that introducing charges at the predicted domain interface in the betaA3 homodimer may contribute to the insolubilization of lens crystallins or favor other, more stable, crystallin subunit interactions.
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Project description:?-Crystallin is a major protein in the human lens that is perceived to help to maintain the transparency of the lens through its chaperone function. In this study, we demonstrate that many lens proteins including ?A-crystallin are acetylated in vivo. We found that K70 and K99 in ?A-crystallin and, K92 and K166 in ?B-crystallin are acetylated in the human lens. To determine the effect of acetylation on the chaperone function and structural changes, ?A-crystallin was acetylated using acetic anhydride. The resulting protein showed strong immunoreactivity against a N(?)-acetyllysine antibody, which was directly related to the degree of acetylation. When compared to the unmodified protein, the chaperone function of the in vitro acetylated ?A-crystallin was higher against three of the four different client proteins tested. Because a lysine (residue 70; K70) in ?A-crystallin is acetylated in vivo, we generated a protein with an acetylation mimic, replacing Lys70 with glutamine (K70Q). The K70Q mutant protein showed increased chaperone function against three client proteins compared to the Wt protein but decreased chaperone function against ?-crystallin. The acetylated protein displayed higher surface hydrophobicity and tryptophan fluorescence, had altered secondary and tertiary structures and displayed decreased thermodynamic stability. Together, our data suggest that acetylation of ?A-crystallin occurs in the human lens and that it affects the chaperone function of the protein.
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Project description:Human age-onset cataracts are believed to be caused by the aggregation of partially unfolded or covalently damaged lens crystallin proteins; however, the exact molecular mechanism remains largely unknown. We have used microseconds of molecular dynamics simulations with explicit solvent to investigate the unfolding process of human lens gammaD-crystallin protein and its isolated domains. A partially unfolded folding intermediate of gammaD-crystallin is detected in simulations with its C-terminal domain (C-td) folded and N-terminal domain (N-td) unstructured, in excellent agreement with biochemical experiments. Our simulations strongly indicate that the stability and the folding mechanism of the N-td are regulated by the interdomain interactions, consistent with experimental observations. A hydrophobic folding core was identified within the C-td that is comprised of a and b strands from the Greek key motif 4, the one near the domain interface. Detailed analyses reveal a surprising non-native surface salt-bridge between Glu135 and Arg142 located at the end of the ab folded hairpin turn playing a critical role in stabilizing the folding core. On the other hand, an in silico single E135A substitution that disrupts this non-native Glu135-Arg142 salt-bridge causes significant destabilization to the folding core of the isolated C-td, which, in turn, induces unfolding of the N-td interface. These findings indicate that certain highly conserved charged residues, that is, Glu135 and Arg142, of gammaD-crystallin are crucial for stabilizing its hydrophobic domain interface in native conformation, and disruption of charges on the gammaD-crystallin surface might lead to unfolding and subsequent aggregation.
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Project description: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.
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Project description:A 3-dimensional model of the human eye lens protein gamma S-crystallin has been constructed using comparative modeling approaches encoded in the program COMPOSER on the basis of the 3-dimensional structure of gamma-crystallin and beta-crystallin. The model is biased toward the monomeric gamma B-crystallin, which is more similar in sequence. Bovine gamma S-crystallin was shown to be monomeric by analytical ultracentrifugation without any tendency to form assemblies up to concentrations in the millimolar range. The connecting peptide between domains was therefore built assuming an intramolecular association as in the monomeric gamma-crystallins. Because the linker has 1 extra residue compared with gamma B and beta B2, the conformation of the connecting peptide was constructed by using a fragment from a protein database. gamma S-crystallin differs from gamma B-crystallin mainly in the interface region between domains. The charged residues are generally paired, although in a different way from both beta- and gamma-crystallins, and may contribute to the different roles of these proteins in the lens.
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Project description:The vertebrate eye lens contains high concentrations of crystallins. The dense lenses of fish are particularly abundant in a class called ?M-crystallin whose members are characterized by an unusually high methionine content and partial loss of the four tryptophan residues conserved in all ?-crystallins from mammals which are proposed to contribute to protection from UV-damage. Here, we present the structure and dynamics of ?M7-crystallin from zebrafish (Danio rerio). The solution structure shares the typical two-domain, four-Greek-key motif arrangement of other ?-crystallins, with the major difference noted in the final loop of the N-terminal domain, spanning residues 65-72. This is likely due to the absence of the conserved tryptophans. Many of the methionine residues are exposed on the surface but are mostly well-ordered and frequently have contacts with aromatic side chains. This may contribute to the specialized surface properties of these proteins that exist under high molecular crowding in the fish lens. NMR relaxation data show increased backbone conformational motions in the loop regions of ?M7 compared to those of mouse ?S-crystallin and show that fast internal motion of the interdomain linker in ?-crystallins correlates with linker length. Unfolding studies monitored by tryptophan fluorescence confirm results from mutant mouse ?S-crystallin and show that unfolding of a ??-crystallin domain likely starts from unfolding of the variable loop containing the more fluorescently quenched tryptophan residue, resulting in a native-like unfolding intermediate.
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Project description:The small heat shock protein ?A-crystallin is a molecular chaperone important for the optical properties of the vertebrate eye lens. It forms heterogeneous oligomeric ensembles. We determined the structures of human ?A-crystallin oligomers by combining cryo-electron microscopy, cross-linking/mass spectrometry, NMR spectroscopy and molecular modeling. The different oligomers can be interconverted by the addition or subtraction of tetramers, leading to mainly 12-, 16- and 20-meric assemblies in which interactions between N-terminal regions are important. Cross-dimer domain-swapping of the C-terminal region is a determinant of ?A-crystallin heterogeneity. Human ?A-crystallin contains two cysteines, which can form an intramolecular disulfide in vivo. Oxidation in vitro requires conformational changes and oligomer dissociation. The oxidized oligomers, which are larger than reduced ?A-crystallin and destabilized against unfolding, are active chaperones and can transfer the disulfide to destabilized substrate proteins. The insight into the structure and function of ?A-crystallin provides a basis for understanding its role in the eye lens.
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Project description: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.
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Project description:Structural impact of the intramolecular disulfide bond on human αA-crystallin
2019-10-07 | PXD013587 | Pride
Project description:The complete sequence of human gamma s-crystallin has been determined and confirmed using a combination of MS methods, peptide sequencing and cDNA sequencing. Regions 21-35 and 102-107, which were previously assumed to be the same as the bovine sequence, differ from the bovine sequence at residues 22, 28, 31 and 104. An additional six residues were also found to be different from the original sequence determined for Pakastani lenses. Whether these differences represent errors in the original sequence or two different sequences among human lens crystallins is not yet known.
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