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At least 19 recordsLinked to original sources
PubMed1995

The expanding small heat-shock protein family, and structure predictions of the conserved "alpha-crystallin domain".

The ever-increasing number of proteins identified as belonging to the family of small heat-shock proteins (shsps) and alpha-crystallins enables us to reassess the phylogeny of this ubiquitous protein family. While the prokaryotic and fungal representatives are not properly resolved, most of the plant and animal shsps and related proteins are clearly grouped in distinct clades, reflecting a history of repeated gene duplications. The members of the shsp family are characterized by the presence of a conserved homologous "alpha-crystallin domain," which sometimes is present in duplicate. Predictions are made of secondary structure and solvent accessibility of this domain, which together with hydropathy profiles and intron positions support the presence of two similar hydrophobic beta-sheet-rich motifs, connected by a hydrophilic alpha-helical region. Together with an overview of the newly characterized members of the shsp family, these data help to define this family as being involved as stable structural proteins and as molecular chaperones during normal development and induced under pathological and stressful conditions.

Amino Acid Sequence↗
PubMed1993

Analysis of the resistance to heat and hydrogen peroxide stresses in COS cells transiently expressing wild type or deletion mutants of the Drosophila 27-kDa heat-shock protein.

The Drosophila melanogaster small heat-shock protein, hsp27 (Dhsp27) belongs to a family of polypeptides which shares a sequence related to alpha-crystallin and which protect cell against heat shock. Dhsp27 accumulates following heat shock and, in absence of stress, in the central nervous system, imaginal discs and the gonads of the developing fly. Two internal and adjacent deletion mutants in the conserved alpha-crystallin domain of Dhsp27 were constructed. Expression vectors containing either the coding sequence of Dhsp27 or that of the two deletion mutants linked to the Simian-Virus-40 late promoter were used to transfect monkey COS cells. The transient expression of Dhsp27 was found to decrease the sensitivity of COS cells to heat and hydrogen-peroxide stresses as judged by Trypan-blue staining and indirect immunofluorescence analysis. Using this rapid test, we observed that a deletion of 62 amino acids, which lies at the 5' end of the conserved alpha-crystallin domain and covers the first 41 amino acids of this region had only a weak effect on the protective activity of Dhsp27. This suggests that the N-terminal half of the conserved alpha-crystallin domain may not be essential for the protective activity of the small hsp. In contrast, Dhsp27 was no more active when the last 42 amino acids of the alpha-crystallin domain were deleted. Biochemical fractionation and indirect immunofluorescence analysis indicated that the protective function of Dhsp27 was localized at the level of the nucleus.

Animals↗
PubMed1994

Elastase inhibition by the C-terminal domains of alpha-crystallin and small heat-shock protein.

alpha-Crystallin, an abundant eye-lens protein and a stress protein in other tissues, shows structural and functional similarities with the small heat-shock proteins. One of the properties in common is the inhibition of elastase. We now report that the separated subunits of alpha-crystallin, alpha A and alpha B, also exhibit elastase inhibition, whereas phosphorylation of these subunits apparently has no influence on the inhibitory capacity. Furthermore, for both alpha A-crystallin and mouse HSP25 the putative C-terminal structural domain, comprising the major region of homology between these proteins, is sufficient to give elastase inhibition. With database search no homology could be found between the three proteins under investigation and any of the known consensus sequences of proteinase inhibitor families.

Amino Acid Sequence↗
PubMed1993

Comparison of the homologous carboxy-terminal domain and tail of alpha-crystallin and small heat shock protein.

The C-terminal domain and tail, which is the most conserved region of the alpha-crystallin/small heat shock protein (HSP) family, was obtained from rat alpha A-crystallin, bovine alpha B-crystallin and mouse HSP25. All three domains have primarily beta-sheet conformation and less than 10% of alpha-helix, like the proteins from which they are derived. Whereas the C-terminal part of alpha A-crystallin forms dimers or tetramers, the corresponding regions of alpha B-crystallin and HSP25 form larger aggregates. The heat-protective activity, recently described for the alpha-crystallin/small HSP family, is not retained in the C-terminal domain and tail. In the course of this study some differences with the previously published sequence of HSP25 were observed, and a revision is proposed.

Amino Acid Sequence↗
PubMed1988

Dogfish alpha-crystallin sequences. Comparison with small heat shock proteins and Schistosoma egg antigen.

The amino acid sequences of the alpha-crystallin A and B chains of the dogfish, Squalus acanthias, have been determined. Comparison with alpha-crystallins from other species reveals that charged amino acid replacements have been strongly avoided in the evolution of this lens protein. The homology of alpha-crystallins with the small heat shock proteins is pronounced throughout the major part of the proteins, starting from the position of the first intron in the alpha-crystallin genes, but is also detectable in the amino-terminal sequences of human, Xenopus, and Drosophila small heat shock proteins. In addition, a remarkable short sequence similarity is present only in the amino termini of dogfish alpha B and Drosophila HSP22. The Schistosoma egg antigen p40 turns out to have a tandemly repeated region of homology with the common sequence domain of alpha-crystallins and small heat shock proteins. Comparison of hydropathy profiles indicates the conservation of conformation of the common domains in these three families of proteins. Construction of phylogenetic trees suggests that the alpha A and alpha B genes apparently originated from a single ancestral small heat shock protein gene and indicates that introns have been lost during the evolution of the heat shock protein genes.

Amino Acid Sequence↗
PubMed2026

Mining the sHSP20 (small heat-shock protein) gene family in finger millet (Eleusine coracana (L.) Gaertn.): structural, evolutionary and predicted abiotic-stress-responsive insights.

Small heat-shock proteins (sHSPs, the HSP20 family) are ATP-independent molecular chaperones that hold partially unfolded substrates and protect the proteome during heat and other abiotic stresses; every member is defined by a conserved &#x3b1;-crystallin domain (ACD). Finger millet (Eleusine coracana) is a climate-resilient, calcium-rich allotetraploid cereal of the semi-arid tropics whose HSP20 repertoire had not been catalogued. The present study is an entirely computational (in silico) analysis of the chromosome-scale reference genome of finger millet (NCBI GenBank assembly GCA_032690845.1, cultivar KNE 796-S). Mining the predicted proteome with the ACD profile (Pfam PF00011) and confirming every candidate by NCBI CD-search recovered 76 non-redundant ACD-bearing HSP20 genes (EcHSP20-1-EcHSP20-76). Based on phylogeny and TargetP-predicted localization, the members were classified into ten subfamilies: seven cytosolic/nuclear classes (C-I to C-VII, 60 members) together with chloroplastic (11), mitochondrial (3) and endoplasmic-reticulum (2) groups. The proteins ranged from 110 to 355 amino acids (12.1-39.2&#xa0;kDa) with theoretical pI of 4.85-9.69. The 76 loci were distributed over 14 of the 18 chromosomes and were conspicuously absent from chromosomes 8&#xa0;A, 8B, 9&#xa0;A and 9B, with pronounced clustering on chromosomes 1, 2, 3 and 6. Duplication analysis detected 149 paralogous pairs (49 homoeologous, 80 segmental/dispersed and 18 tandem); 147 of 148 pairs for which substitution rates could be calculated returned Ka/Ks&#x2009;<&#x2009;1 (mean 0.20), indicating strong purifying selection consistent with retention after whole-genome/allopolyploid duplication. Promoter analysis (PlantCARE) revealed enrichment of abscisic-acid-responsive (ABRE), MYB/MYC drought-related, STRE, DRE, low-temperature (LTR) and methyl-jasmonate/salicylic-acid elements, whereas canonical heat-shock elements (HSE) were not recovered. Expression profiling against a public drought transcriptome (SRP081350) showed that about half of the genes (39 of 76) are transcribed in leaf tissue, the expressed fraction being dominated by the cytosolic class C-I. This first finger-millet HSP20 catalogue provides a verified, reproducible framework and nominates computationally predicted candidate genes for future functional work on thermotolerance in cereals.

Allotetraploid↗
PubMed1991

Micellar subunit assembly in a three-layer model of oligomeric alpha-crystallin.

Differential scanning calorimetry was performed to monitor the heat-induced changes that occur in the structural domain of lens alpha-crystallin. Circular dichroism and fluorescence also were used to resolve the controversial issue of the quaternary structure of alpha-crystallin. Based on the thermal behavior as monitored by these techniques, a model is proposed that can account for all previous data as well as the currently reported thermal data. The proposed model of native alpha-crystallin has a three-layer structure in which the inner layer (core) is a micelle containing 12 subunits arranged in cuboctahedral symmetry. The apolar region is directed inward constituting a hydrophobic core similar to a micelle and adding structural stability. A second layer of six subunits has a similar but not identical structure to the first layer, directing its apolar face toward the hydrophobic core. Thus, these two layers constitute a micelle-like structure with octahedral symmetry. The third layer adds more subunits for a total of not more than 24. Differential scanning calorimetry, circular dichroism, and fluorescence studies indicated that the inner two-layer structure of molecular mass 360 kDa is highly stable and is most likely of the alpha m form. The three-layer structure of the native protein, however, is rather unstable. At 35-45 degrees C the outer layer dissociates from the inner two layers, and at higher temperatures rapidly reassociates to a slightly modified two-layer structure with a stability similar to that of alpha m. The proposed model does not require any specific assembly of the alpha A and alpha B subunits in each layer, but the fluorescence results suggest that the native inner two layers probably contain mostly alpha A.

Animals↗
PubMed1992

Structural organization and stability of a thermoresistant domain generated by in vivo hydrolysis of the alpha-crystallin B chain from calf lens.

A protein fragment (M(r) approximately 9000) isolated from the cortex of nonpathological calf lenses has been structurally characterized. The polypeptide structure was well organized (39% alpha-helix, 33% beta-structure, and 28% remainder) according to the far-ultraviolet circular dichroism. The fluorescence was heterogeneous for the presence of two tryptophan classes. Structure perturbation by pH and denaturant revealed cooperative structural transitions which are characteristics of a globular organization. A single-step unfolding curve induced by Gdn-HCl (midpoint = 1.38 M Gdn-HCl) was monitored by emission maximum shift as well as by far-ultraviolet circular dichroism. This transition was analyzed as a two-state process. The standard free energy of unfolding in the absence of the denaturant, delta Go (H2O), was found to be 10.80 +/- 0.25 kJ/mol at 20 degrees C and pH 7.4. The fragment also shows an unusual thermal resistance. Its structure was unperturbed up to 90 degrees C according to the fluorescence and dichroism. This last property, its peculiar amino acid composition, and the sequence of a small segment are shared, among crystallins, only with the N-terminal region of the alpha-crystallin B chain. A search for proteolysis sites along the alpha-crystallin B chain sequence revealed that it possesses specific points for proteinase attack. These sites are particularly exposed and clustered in a very flexible region in the middle of the protein sequence. They are also well represented in the C-terminal extension of the molecule while a few are buried in the N-terminal region.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

High-molecular-weight protein aggregates of calf and cow lens: spectroscopic evaluation.

To gain insight into the molecular features of the high-molecular-weight (HMW) fraction of soluble lens proteins and their changes in aging, we isolated this fraction from the nucleus of calf and cow lenses and measured fluorescence and circular dichroism (CD) properties of the samples. Not only was there an increase in the HMW fraction in the older lens, but there was also an age-related difference in tertiary structure that was clearly manifested in the fluorescence and CD parameters. The far-u.v. CD of low- and high-molecular-weight proteins do not differ significantly in band position and magnitude, but the near-u.v. CD of HMW protein does differ distinctly from that of all other crystallins (alpha, beta and gamma); the entire CD spectrum of this protein is displayed in the negative region. Millipore filtration further revealed that HMW aggregates are essentially a polydisperse population of different conformation (tertiary structure) and that these aggregates are associated by non-convalent interactions. This association is caused mainly by the apolar (hydrophobic) nature of the constituent protein. alpha-Crystallin has more hydrophobic domain along the peptide chain that do other crystallins and thus is likely to be the predominant protein in HMW aggregates.

Aging↗
PubMed1995

1H NMR spectroscopy reveals that mouse Hsp25 has a flexible C-terminal extension of 18 amino acids.

The small heat-shock proteins (Hsps) exist as large aggregates and function by interacting and stabilising non-native proteins in a chaperone-like manner. Two-dimensional 1H NMR spectroscopy of mouse Hsp25 reveals that the last 18 amino acids have great flexibility with motion that is essentially independent of the domain core of the protein. The lens protein, alpha-crystallin, is homologous to Hsp25 and its two subunits also have flexible C-terminal extensions. The flexible region in Hsp25 encompasses exactly that expected from sequence comparison with alpha-crystallin implying that both proteins have similar structures and that the C-terminal extensions could be of functional importance for both proteins.

Amino Acid Sequence↗
PubMed1992

Expression and aggregation of recombinant alpha A-crystallin and its two domains.

The 20 kDa alpha A and alpha B subunits of alpha-crystallin from mammalian eye lenses form large aggregates with an average molecular weight of 800,000. To get insight into the interactions responsible for aggregate formation, we expressed in Escherichia coli the putative N- and C-terminal domains of alpha A-crystallin, as well as the intact alpha A-crystallin chain. The proteins are expressed in a stable form and in relatively high amounts (20-60% of total protein). Recombinant alpha A-crystallin and the C-terminal domain are expressed in a water-soluble form. Recombinant alpha A-crystallin forms aggregates comparable with alpha-crystallin aggregates from calf lenses, whereas the C-terminal domain forms dimers or tetramers. The N-terminal domain is expressed in an initially water-insoluble form. After solubilization, denaturation and reaggregation the N-terminal domain exists in a high molecular weight multimeric form. These observations suggest that the interactions leading to aggregation of alpha A-crystallin subunits are mainly located in the N-terminal half of the chain.

Base Sequence↗
PubMed1990

The alternative splicing product alpha Ains-crystallin is structurally equivalent to alpha A and alpha B subunits in the rat alpha-crystallin aggregate.

In rodents and some other unrelated mammals, alternative splicing of the alpha A-crystallin gene transcript results in the synthesis of the elongated alpha Ains-crystallin chain. This polypeptide is identical to the normal alpha A-crystallin chain of 173 residues, but contains an additional sequence of 23 amino acid residues inserted between positions 63 and 64. To determine the effects of this insert peptide, the structure of the rat alpha-crystallin aggregate and its subunits alpha A-, alpha Ains- and alpha B-crystallin was studied using fluorescence spectra, partial urea dissociation, and lactoperoxidase-catalysed iodination of surface residues. The data suggest that all alpha-crystallin subunits occupy equivalent positions in the protein aggregate, and that the insert peptide merely elongates the connecting peptide between the putative amino- and carboxyl-terminal domain of the alpha A-crystallin subunit.

Amino Acid Sequence↗
PubMed1993

Evolution of the alpha-crystallin/small heat-shock protein family.

The common characteristic of the alpha-crystallin/small heat-shock protein family is the presence of a conserved homologous sequence of 90-100 residues. Apart from the vertebrate lens proteins--alpha A- and alpha B-crystallin--and the ubiquitous group of 15-30-kDa heat-shock proteins, this family also includes two mycobacterial surface antigens and a major egg antigen of Schistosoma mansoni. Multiple small heat-shock proteins are especially present in higher plants, where they can be distinguished in at least two classes of cytoplasmic proteins and a chloroplast-located class. The alpha-crystallins have recently been found in many tissues outside the lens, and alpha B-crystallin, in particular, behaves in many respects like a small heat-shock protein. The homologous sequences constitute the C-terminal halves of the proteins and probably represent a structural domain with a more variable C-terminal extension. These domains must be responsible for the common structural and functional properties of this protein family. Analysis of the phylogenetic tree and comparison of the biological properties of the various proteins in this family suggest the following scenario for its evolution: The primordial role of the small heat-shock protein family must have been to cope with the destabilizing effects of stressful conditions on cellular integrity. The alpha-crystallin-like domain appears to be very stable, which makes it suitable both as a surface antigen in parasitic organisms and as a long-living lens protein in vertebrates. It has recently been demonstrated that, like the other heat-shock proteins, the alpha-crystallins and small heat-shock proteins function as molecular chaperones, preventing undesired protein-protein interactions and assisting in refolding of denatured proteins. Many of the small heat-shock proteins are differentially expressed during normal development, and there is good evidence that they are involved in cytomorphological reorganizations and in degenerative diseases. In conjunction with the stabilizing, thermoprotective role of alpha-crystallins and small heat-shock proteins, they may also be involved in signal transduction. The reversible phosphorylation of these proteins appears to be important in this respect.

Amino Acid Sequence↗
PubMed1994

A 1H NMR spectroscopic comparison of gamma S- and gamma B-crystallins.

Two-dimensional 1H NMR spectroscopic studies are presented on bovine gamma S- and gamma B-crystallin. In gamma S-crystallin, the four N-terminal residues have great flexibility compared with the rest of the molecule and assume a random coil conformation. NMR spectroscopy and electrospray mass spectrometry show that the N-terminal residue is acetylated. Thus, gamma S-crystallin is similar to the acidic beta-crystallins in having a flexible N-terminal extension and an N-terminus that is blocked with an acetyl group but no C-terminal extension. In addition to the short N-terminal extension in gamma S-crystallin, other unassigned resonances are also observed in the NMR spectra. In gamma B-crystallin, however, cross-peaks in the NH to alpha-CH region of the spectrum are essentially restricted to the last three residues of the C-terminal domain. The NMR data imply that gamma S-crystallin has a more flexible structure than gamma B-crystallin. Sedimentation equilibrium studies on gamma S-crystallin are consistent with this proposal. Resonances from the N-terminal extension of gamma S-crystallin are not affected by the presence of alpha-crystallin implying that this region is not involved in interactions between the two molecules. It is concluded that gamma S-crystallin shares structural properties which are intermediate between the beta- and gamma-crystallins.

Animals↗
PubMed1983

Structural homology of lens crystallins. III. Secondary structure estimation from circular dichroism and prediction from amino acid sequences.

Circular dichroism spectra (196-240 nm) of calf alpha-, beta H-, beta L- and gamma-crystallins were measured and analyzed over the entire wavelength range with five curve-fitting procedures for estimating protein secondary structure. For gamma-crystallin the estimates are in good agreement with the X-ray structure. For all four crystallins the estimates are very similar: 0-9% alpha-helix and 51-68% beta-sheet. This is in accordance with the three-dimensional homology of beta Bp- and gamma 2-crystallin polypeptide chains as postulated from their 30% sequence homology, and suggests that alpha A- and alpha B-crystallin chains may also have a corresponding structure. Secondary structure elements in the four amino acid sequences were predicted using two different comprehensive prediction methods. For gamma 2-crystallin the predictions of beta-sheet are in good agreement with the X-ray structure and with circular dichroism estimates. For beta Bp-crystallin only the C-terminal domain secondary structure predictions are considered satisfactory, which possibly relates to the proposed role of the N-terminal domain in subunit interactions. The combined predictions for alpha A- and alpha B-chains (3% helix, 49% sheet) are in excellent agreement with circular dichroism. Moreover, the good alignment of predicted beta-sheet segments in alpha-crystallin chains with known beta-sheet strands in gamma 2- (and presumably beta Bp-) crystallin strongly supports a similar 4-motif folding pattern in all four calf crystallin chains.

Amino Acid Sequence↗
PubMed1985

Spectroscopic studies on human lens crystallins.

Human lens crystallins were studied by absorption, circular dichroism and fluorescence spectroscopy. The absorption spectra in the near-ultraviolet region show some differences in intensity, but spectral features are similar, except for the alpha-crystallin, which gives a fine structure due to phenylalanine between 250 and 270 nm. Tryptophan fluorescence and near-ultraviolet circular dichroism indicate that tryptophan residues are more exposed in alpha-crystallin than in either beta- or gamma-crystallin, and that the degree of exposure decreases in the order of alpha less than beta 1 greater than beta 2 greater than beta 3 greater than gamma. The far ultraviolet CD suggests that these proteins exist mainly in a beta-sheet conformation and that the amount does not vary much among them. The greater exposure of the tryptophan residues in the high-molecular-weight crystallins may reflect greater unfolding in their protein domains. Spectroscopic measurements are thus useful in predicting protein tertiary structure in the absence of the complete sequence and X-ray data. The fact that the high-molecular-weight proteins exist in a more unfolded state may render them more vulnerable to exogeneous insults, and these effects may be studied by spectroscopic measurements.

Chromatography, Gel↗
PubMed1989

Folding-unfolding and aggregation-dissociation of bovine alpha-crystallin subunits; evidence for unfolding intermediates of the alpha A subunits.

The aggregation and dissociation behavior of bovine alpha-crystallin as well as the folding and unfolding of its subunits were investigated by equilibrium studies using tryptophan fluorescence measurements and two isoelectric focusing techniques, viz. isoelectric focusing across a urea gradient and isoelectric focusing in two dimensions with different concentrations of urea. It was found that the alpha B chains lose their ability to aggregate and start unfolding at a lower concentration of urea than the alpha A chains. Equilibrium intermediates were found upon unfolding or refolding of alpha A subunits, which can be explained by a two-domain organization of these molecules.

Animals↗