Eye-lens proteins: The three-dimensional structure of β-crystallin predicted from monomeric γ-crystallin (original) (raw)
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Phosphorylation of @-Crystallin B 2 ( @ Bp ) in the Bovine Lens *
2001
Three major “P-labeled polypeptides were found in the soluble fraction of bovine lenses cultured in a medium containing [32P]orthophosphate. Two of the polypeptides corresponded to the phosphorylated A and B chains of a-crystallin. In this communication, the third polypeptide is now identified. This polypeptide is characterized by a molecular weight of 27,000 and a PI of 6.6, eluted exclusively in the j 3 ~ ~ ~ fraction of a CL-GB gel filtration separation of lens soluble material, and could be further purified by DE52 anion exchange chromatography. The only “P-labeled amino acid detected was phosphoserine. A single 32P-labeled peptide was observed after tryptic digestion and two-dimensional mapping. The amino acid sequence of the purified peptide is Gly-Ala-Phe-His-Pro-Ser-Ser. This sequence exactly matches the expected C-terminal tryptic fragment, residues 198-204, of the bovine Bcrystallin B2. The results of carboxypeptidase A digestion of the 32P-labeled peptide suggest that onl...
Biochemical and Biophysical Research Communications, 1970
The vertebrate lens is composed of two distinct cell types, the epithelial cell and the fiber cell. ol-Crystallin, a structural protein found in both cell types, is composed of four subunits @Ai, olA2, aB1, and olB2). In the epithelial cells the cr-crystallin consists mainly of crA2 and crB2 with trace amounts of crA1 and cyB1. In the fiber cell there is a large increase in the amount of c~A1 and olBl subunits. This quantitative increase in two specific subunits is related to the process of cellular growth and differentiation in the vertebrate lens.
Multiple genes coding for the frog eye lens γ-crystallins
Gene, 1984
Three new recombinant cDNA clones coding for y-crystallins have been identified in the frog (Rana temporaria) clonotheque by hybrid-selected translation/immunoprecipitation experiments, in addition to the y-1-crystallin clone that was isolated and sequenced previously [Tomarev et al., Gene 17 (1982) 131-138; FEBS Letters 146 (1982) 315-3181. mRNA species coding for all these y-crystallins are about 650 nucleotides in length, but differ in structure, as follows from restriction and sequence analysis of the cloned cDNAs. The conclusion is that the R. temporaria genome contains a family of at least four similar but not identical y-crystallin genes. The complete nucleotide sequence has been determined for the cDNA of one of these clones coding for y-2-crystallin. It is 69% homologous with that of R. temporaria y-1-crystallin and contains four regions of partial internal homology corresponding to the four structural folding units of the y-crystallin molecules. An unusual feature of the y-Zcrystallin amino acid sequence is the high lysine/arginine ratio equal to 1.1, in contrast to 0.05-0.16 for other known y-crystallins.
Classification of Rat Lens Crystallins and Identification of Proteins Encoded by Rat Lens mRNA
European Journal of Biochemistry, 2005
Endogenous rat lens crystallins have been separated by gel filtration into four fractions, a, fiH, Br. and 7-crystallin. Elution patterns of soluble lens proteins from animals of different ages show a relative decrease of /?H and ;-crystallin during aging. Conversely the relative amounts of r and jL-crystallin are enhanced. The rat crystallin subunits from the four fractions were characterized by one-dimensional and two-dimensional gel electrophoretic techniques. From the results a classification could be derived and a nomenclature for the soluble rat lens proteins is proposed. The products synthesized by rat lens mRNAs in a heterologous cell-free system have also been characterized. Co-electrophoresis of the radioactive products synthesized de novo together with the isolated unlabeled protein fractions on two-dimensional gels shows the relation between primary gene products and their posttranslationally modified derivatives.
Biochemical and Biophysical Research Communications, 1974
incorporation of ~3H3 leucine into ollgomeric a-crystallin via individual subunits has beenmeasured in epithelial cells and cortex fiber cells from adult bovine lenses in vitro. Our data show that the ratio of [3H~ leucine incorporation via subunlts aB2 and aA 2 Ts shifted from a value of about 1:2 in epithelial cells to a value of about 1:3 in fiber cells. Thus, in this system, cellular differentiation is accompanied by a change in the stolchiometry of assembly of individual subunits to form the oligomerlc a-crystallin molecule. These results indicate possible changes in the rates of synthesis of individual a-crystallln subunits. a-Crystallin is an ollgomerlc structural protein that is found in both epithelial cells and fiber cells of the bovine lens (1) and has a molecular weight of approximately 1 X 106 (2, 3). Treatment with urea and mercaptoethanol dissociates the oligomer into its polypeptide subunits, all of which have single molecular weights of about 25,000 (4). These subunits can be resolved into two acidic proteins (aA 1 and aA2) , which have isoelectric points of 5.6 and 5.9, respectively, and two basic proteins (aB 1 and aB2) , which have isoelectric points *Research sponsored by the U. S. Atomic Energy Commission under contract with the Union Carbide Corporation.
Journal of Molecular Biology, 2000
bg-crystallins from the eye lens are proteins consisting of two similar domains joined by a short linker. All three-dimensional structures of native proteins solved so far reveal similar pseudo-2-fold pairing of the domains re¯ecting their presumed ancient origin from a single-domain homodimer. However, studies of engineered single domains of members of the bg-crystallin superfamily have not revealed a prototype ancestral solution homodimer. Here we report the 2.35 A Ê X-ray structure of the homodimer of the N-terminal domain of rat bB2-crystallin (bB2-N). The two identical domains pair in a symmetrical manner very similar to that observed in native bg-crystallins, where N and C-terminal domains (which share $35% sequence identity) are related by a pseudo-2-fold axis. bB2-N thus resembles the ancestral prototype of the bg-crystallin superfamily as it self-associates in solution to form a dimer with an essentially identical domain interface as that between the N and C domains in bg-crystallins, but without the bene®t of a covalent linker. The structure provides further evidence for the role of two-domain pairing in stabilising the protomer fold. These results support the view that the bg-crystallin superfamily has evolved by a series of gene duplication and fusion events from a single-domain ancestor capable of forming homodimers.
Biochemistry, 2009
The human lens proteins β-crystallins are subdivided into acidic (βA1-βA4) and basic (βB1-βB3) subunit groups. These structural proteins exist at extremely high concentrations and associate into oligomers under physiological conditions. Crystallin acidic-basic pairs tend to form strong heteromolecular associations. The long N-terminal extensions of β-crystallins may influence both homo-and heteromolecular interactions. However, identification of the critical regions of the extensions mediating protein associations have not been previously addressed. This was studied by comparing the self association and heteromolecular associations of wild-type recombinant βA3 and βB1 crystallins and their N-terminal truncated counterparts (βA3ΔN30 and βB1ΔN56) using several biophysical techniques including analytical ultracentrifugation and fluorescence spectroscopy. Removal of the N-terminal extension of βA3 had no effect on dimerization or heteromolecular tetramer formation with βB1. In contrast, the self association of βB1ΔN56 increased resulting in homotetramer formation and heteromolecular association with βA3 was blocked. Limited proteolysis of βB1 produced βB1ΔN47, which similar to intact protein formed dimers but in contrast showed enhanced heteromolecular tetramer formation with βA3. The tryptic digestion was of physiological significance, corresponding to protease processing sites observed in-vivo. Molecular modeling of the N-terminal βB1 extension indicates structural features which position a mobile loop in the vicinity of these processing sites. The loop is derived from residues 48-56 which appear critical for mediating protein interactions with βA3-crystallin. Keywords β-crystallins; N-terminal extension; protein association Crystallins are the major structural proteins of the lens, where at very high concentrations they are responsible for the transparency and high refractive index (1). In the mammalian lenses, crystallins can be grouped in two families, small heat shock-related α-crystallins and βγcrystallins (1;2). All βγ-crystallins are composed of one, two or multiple βγ-crystallin domains, where each domain is made up of two β-stranded Greek-key motifs. The β-crystallins are distinguished from γ crystallins by having either N-and C-termini extensions, for basic βcrystallins (βB1, βB2, βB3), or only an N-terminal extension, for acidic forms (βA1/A3, βA2, βA4) (3;4). Whereas γ crystallins exist as monomers, β-crystallins are known to associate into
RECONSTRUCTING NORMAL a-CRYSTALLIN FROM THE MODIFIED CATARACTOUS PROTEIN
"-Crystallin from human cataractous lenses is so extensively modified thai il is unsuitable for structural studies. We have disassembled the protein and, from the remaining traces of unmodified polypepiides, reconstructed a more homogeneous and conformationally discrete molecule. We suggest thai this approach may be particularly useful to studies of enzymology and molecular gerontology.