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C Slingsby

Publications and source records attributed to C Slingsby.

At least 19 recordsLinked to original sources
PubMed1992

Protein interactions in the calf eye lens: interactions between beta-crystallins are repulsive whereas in gamma-crystallins they are attractive.

Non-specific interactions in beta- and gamma-crystallins have been studied by solution X-ray scattering and osmotic pressure experiments. Measurements were carried out as a function of protein concentration at two ionic strengths. The effect of temperature was tested between 7 degrees C and 31 degrees C. Two types of interactions were observed. With beta-crystallin solutions, a repulsive coulombic interaction could be inferred from the decrease of the normalized X-ray scattering intensity near the origin with increasing protein concentration and from the fact that the osmotic pressure increases much more rapidly than in the ideal case. As was previously observed with alpha-crystallins, such behaviour is dependent upon ionic strength but is hardly affected by temperature. In contrast, with gamma-crystallin solutions, the normalized X-ray scattering intensity near the origin increases with increasing protein concentration and the osmotic pressure increases less rapidly than in the ideal case. Such behaviour indicates that attractive forces are predominant, although we do not yet know their molecular origin. Under our experimental conditions, the effect of temperature was striking whereas no obvious contribution of the ionic strength could be seen, perhaps owing to masking by the large temperature effect. The relevance of the different types of non-specific interactions for lens function is discussed.

Animals↗
PubMed1992

Structural studies on beta H-crystallin from bovine eye lens.

Bovine lens beta H-crystallin, isolated at pH 6.7, undergoes reversible dissociation into dimers and an intermediate size of oligomer (peak A) at pH 5.4. Peak A is enriched in the beta B1 subunit but lacks beta B2, whereas beta B2 is a major component of the dimers. A method for isolation of beta B1 from peak A is described. The pH dependence of the dissociation-reassociation suggests that histidines on the surface of the dimers become buried in the assembly of beta H-crystallin. The positions of the four histidines on the surface of the compact domains of each subunit of the beta B2 homodimer are shown. The beta B1-enriched oligomer has a much lower solubility compared with the beta B2 containing beta H-crystallin. It is possible that beta B2 plays a role in solubilizing beta-crystallin aggregates.

Animals↗
PubMed1991

High resolution structure of an oligomeric eye lens beta-crystallin. Loops, arches, linkers and interfaces in beta B2 dimer compared to a monomeric gamma-crystallin.

beta-Crystallins are polydisperse, oligomeric structural proteins that have a major role in forming the high refractive index of the eye lens. Using single crystal X-ray crystallography with molecular replacement, the structure of beta B2 dimer has been solved at 2.1 A resolution. Each subunit comprises an N and C-terminal domain that are very similar and each domain is formed from two similar "Greek key" motifs related by a local dyad. Sequence differences in the internally quadruplicated molecules, analysed in terms of their beta-sheets, hairpins and arches, give rise to structural differences in the motifs. Whereas the related family of gamma-crystallins are monomers, beta-crystallins are always oligomers. In the beta B2 subunit, the domains, each comprising two motifs, are separated by an extended linking peptide. A crystallographic 2-fold axis relates the two subunits of the dimer so that the N-terminal domain of one subunit of beta B2 and the C-terminal domain of the symmetry-related subunit are topologically equivalent to the two covalently connected domains of gamma B-crystallin. The intersubunit domain interface is very similar to the intradomain interface of gamma B, although many sequence differences have resulted in an increase in polar interactions between domains in beta B2. Comparison of the structures of beta B2 and gamma B-crystallins shows that the two families differ largely in the conformation of their connecting peptides. A further extensive lattice contact indicates a tetramer with 222 symmetry. The ways in which insertions and extensions in the beta-crystallin effect oligomer interactions are described. The two kinds of crystallin are analysed for structural features that account for their different stabilities. These studies are a basis for understanding formation of higher aggregates in the lens.

Amino Acid Sequence↗
PubMed1991

Structure of oligomeric beta B2-crystallin: an application of the T2 translation function to an asymmetric unit containing two dimers.

The molecular structure of the main subunit of the beta-crystallins, components of the vertebrate eye lens, has recently been solved by molecular replacement at 2.1 A resolution [Bax, Lapatto, Nalini, Driessen, Lindley, Mahadevan, Blundell & Slingsby (1990). Nature (London), 347, 776-780]. The protein, beta B2, is a dimer in solution, but a tetramer in the crystal with one subunit in the asymmetric unit of space group I222. Using the crystallographic dimer from this I-centred form the structure of a C222 crystal form of the beta B2 protein with four subunits in the asymmetric unit has now been solved by molecular replacement at 3.3 A. The solution involved the use of a new translation function for non-crystallographic symmetry, based on the T2 function of Crowther & Blow [Acta Cryst. (1967), 23, 544-548].

Crystallins↗
PubMed1991

Isolation and characterization of cDNAs encoding beta A2- and beta A4-crystallins: heterologous interactions in the predicted beta A4-beta B2 heterodimer.

Except for the two acidic chains, beta A2 and beta A4, the primary structures of all bovine beta-crystallins have previously been elucidated, either by direct protein sequencing or prediction from cDNA sequencing. Both beta A2 and beta A4 were found to be synthesized in half-year-old calf lenses and are therefore likely to be present in a cDNA bovine library constructed from mRNA isolated from lenses of that age. A large number of cDNA clones was screened with all available crystallin, actin, vimentin and lens membrane protein MP26 probes and finally with a randomly primed mRNA probe. Clones positive for the latter, but negative for known lens proteins, were isolated and sequenced. beta A2, comprising 197 aa, and beta A4, comprising 209 aa, were identified. Both proteins have a conserved two-domain structure and an N-terminal extension which is variable. A three-dimensional model of the structure of beta A4 was made based on the coordinates of one subunit from the beta B2 dimer which has recently been solved using x-ray diffraction techniques. The resulting heterodimer structure, together with the compiled bovine beta-crystallin sequences, was used to indicate those regions of the sequences which distinguish acidic from basic beta-crystallins with a view to defining structural features necessary for subunit recognition in beta-crystallin aggregates. With the aid of the present data, the complete evolutionary tree of the bovine beta-crystallin family has been constructed, which confirms the early separation of the genes encoding the three acidic and the three basic beta-crystallins.

Amino Acid Sequence↗
PubMed1990

X-ray analysis of beta B2-crystallin and evolution of oligomeric lens proteins.

The beta, gamma-crystallins form a class of homologous proteins in the eye lens. Each gamma-crystallin comprises four topologically equivalent, Greek key motifs; pairs of motifs are organized around a local dyad to give domains and two similar domains are in turn related by a further local dyad. Sequence comparisons and model building predicted that hetero-oligomeric beta-crystallins also had internally quadruplicated subunits, but with extensions at the N and C termini, indicating that beta, gamma-crystallins evolved in two duplication steps from an ancestral protein folded as a Greek key. We report here the X-ray analysis at 2.1 A resolution of beta B2-crystallin homodimer which shows that the connecting peptide is extended and the two domains separated in a way quite unlike gamma-crystallin. Domain interactions analogous to those within monomeric gamma-crystallin are intermolecular and related by a crystallographic dyad in the beta B2-crystallin dimer. This shows how oligomers can evolve by conserving an interface rather than connectivity. A further interaction between dimers suggests a model for more complex aggregates of beta-crystallin in the lens.

Amino Acid Sequence↗
PubMed1990

Quaternary interactions in eye lens beta-crystallins: basic and acidic subunits of beta-crystallins favor heterologous association.

beta-Crystallins are complex eye lens proteins made up of several related basic and acidic subunits that combine to form differently sized oligomers each displaying extensive polydispersity. As the sequences are homologous to the X-ray-determined bilobal structure of gamma-crystallin, beta-subunits are visualized as having a similar structure with additional N- and C-terminal extensions. Two basic (beta B2 and beta B3) and two acidic (beta A3 and beta A4) subunits have been isolated in deaggregating media, refolded, and reassociated in various combinations to determine which components favor dimers or higher oligomers. Homopolymers were compared with beta B2 homodimer in terms of charge, using Mono Q fast protein liquid chromatography, and size, using Superose 12 chromatography. Heterooligomeric formations were monitored by their intermediate charge properties compared with homooligomers. beta B2 associates with either beta B3- or beta A4-forming heterodimers whereas a larger oligomer is formed with beta A3. Naturally occurring beta-crystallin oligomers were analyzed by Mono Q chromatography and PhastGel electrophoresis. Whereas beta B2, beta B3, and beta A4 can each be reassociated to homodimers, beta A4 dimers are not found in native beta-crystallins. beta B2-beta A3 is a major component of intermediate-sized beta L1-crystallin and is absent from dimeric beta L2-crystallin. It is suggested that the pH dependence of the size of beta L1-crystallin is due to a dimer to tetramer equilibrium. By following dimer interactions using Superose 12 chromatography, beta B2-beta A4 was shown to interact with beta B2-beta A3. A model of beta-crystallin structure is proposed based on beta-subunits forming dimers with the next level of organization requiring an acidic subunit, beta A3, with a long N-terminal extension.

Animals↗
PubMed1990

Rapid separation of bovine beta-crystallin subunits beta B1, beta B2, beta B3, beta A3 and beta A4.

Bovine beta-crystallin aggregates, beta H-, beta L1- and beta L2-crystallins, prepared by rapid gel filtration, are each subjected to anion-exchange chromatography in deaggregating media using a Pharmacia Fast Protein Liquid Chromatography System. beta B1, beta B2 and beta A4 subunits are rapidly isolated using a one step Mono Q column from beta H-, beta L2- and beta L1-crystallin, respectively. beta B3 and beta A3 are separated from each other using a second Mono Q column starting from beta L2- and beta L1-crystallin respectively. Whereas beta B2, beta B3 and beta A4 are common to all sizes of aggregate, beta B1 is restricted to beta H-crystallin and beta A3 is absent from beta L2-crystallin.

Animals↗
PubMed1989

Crystallization of a new form of the eye lens protein beta B2-crystallin.

A new crystal form of the bovine oligomeric lens protein beta B2 has been grown in the presence of calcium acetate. The crystals are orthorhombic, I222 or I2(1)2(1)2(1), with cell dimensions a = 77.8 A, b = 83.6 A, c = 109.2 A. This new crystal form, which diffracts to at least 2.5 A, has a and b cell dimensions that are half those of the original crystal form, although there is no simple relationship between the c cell dimensions. The new crystal form reported here contains only one subunit per asymmetric unit, indicating that the dimer lies on a crystallographic 2-fold axis, and is a suitable candidate for molecular replacement studies.

Animals↗
PubMed1989

Packing interactions in the eye-lens. Structural analysis, internal symmetry and lattice interactions of bovine gamma IVa-crystallin.

gamma-Crystallins are a family of low molecular weight proteins found in high concentration in the densely packed regions of high refractive index in vertebrate lenses. Certain members have the characteristic property of a high critical temperature (tc) for phase separation. We report the three-dimensional structure determination of such a protein, bovine lens gamma IVa-crystallin, which has been refined to give an X-ray R-factor of 0.143. Its high tc contrasts with the low tc gamma II-crystallin, whose structure we have already published. The root mean square difference between the alpha-carbon atoms of these two proteins is 0.70 A and gamma IVa has an internal symmetry even higher than that of gamma II. The presence of a protein that exhibits the phenomenon of phase separation at body temperature renders the lens very susceptible to a transformation from transparent to an opaque state due to irregularities in the refractive index. Protein interactions of gamma IVa-crystallin have implications for the mechanism of cataract formation. Modes of self-association behaviour of gamma IVa-crystallin have been inferred from an analysis of the lattice interactions in the crystalline state, where the protein packing density is similar to that of the intact lens. It appears that the point mutation at position 103 from a serine residue in gamma II to a valine in gamma IVa gives rise to a lattice contact formed by two four-stranded beta-sheets in gamma IVa. A group-specific mutation at position 118 from leucine to phenylalanine induces subtle differences in core packing, leading to a reorganization around residue 103. However, the final phase separation determinant may be a complex combination of many side-chain functions.

Amino Acid Sequence↗
PubMed1989

Raman spectroscopy of calf lens gamma-II crystallin: direct evidence for the formation of mixed disulfide bonds with 2-mercaptoethanol and glutathione.

This study presents Raman spectra of calf lens gamma-II crystallin and its reaction products with reduced glutathione, 2-mercaptoethanol and p-hydroxymercuribenzoate. The absence of a disulfide vibration in gamma-III crystallin (both in aqueous solution and in lyophilized state) indicates that the seven thiol groups in this protein are resistant to air oxidation, and are capable of maintaining their reduced state in the absence of added reducing agents during isolation. However, treatment of the protein with low molecular weight thiols such as glutathione and 2-mercaptoethanol results in mixed disulfide bonds. We have detected, for the first time, the S--S bond stretching vibration from the mixed disulfides at 510 cm-1, which is very similar to the 508 cm-1 reported for the inter/intramolecular disulfide bonds in intact mouse lenses (Yu, N.-T., DeNagel, D.C., Pruett, P.L. and Kuck, J.F.R., Jr. (1985). Proc. Natl. Acad. Sci. U.S.A., 82, 7965-8). Upon titration with five equivalents of p-hydroxymercuribenzoate, a strong Raman line was detected at 345 cm-1, which is tentatively attributed to the Hg--S stretching vibration of the mercaptide complex. The S--H vibration region (2500-2700 cm-1) exhibits two resolved peaks at 2562 and 2580 cm-1 with an intensity ratio of 2:5. Both reactive surface thiol groups and buried cysteines give rise to the S--H vibration at 2580 cm-1.

Animals↗
PubMed1988

The use of pseudosymmetry in the rotation function of gamma IVa-crystallin.

Bovine lens gamma IVa-crystallin crystallizes in space group C222(1) with cell dimensions a = 35.1, b = 46.2, c = 186.2 A, and contains one molecule in the asymmetric unit. The structure was determined at 3.0 A resolution using cross-rotation functions and R-factor searches with the bovine lens protein gamma II-crystallin as the model structure. The rotation function appears to be very sensitive to the resolution range and type of coefficient employed; the use of normalized structure-factor amplitudes gave the best results. The potential problem of a pseudo solution due to an internal pseudo-twofold axis was put to advantage by aligning this axis parallel to z. The results of the R-factor search were well defined. The molecular replacement solution was improved by rigid-body least-squares refinement, initially of the whole molecule, then for the two domains. The R factor at this stage was 39.4% at 2.3-10.0 A. The gamma IVa structure has an even higher internal symmetry than gamma II, since the two domains are related by a rotation around the pseudo-twofold axis of 178.7 degrees as compared with 176.2 degrees for gamma II.

Animals↗
PubMed1988

Surface interactions of gamma-crystallins in the crystal medium in relation to their association in the eye lens.

A comparative study of intermolecular interactions in crystals of two homologous low molecular weight proteins, gamma-II and gamma-IIIb crystallins, from calf eye lens was carried out. Crystal packings for these proteins are very different: intermolecular contact areas compose about 33% of the total accessible surface area of gamma-II as compared with 13% in gamma-III. Two key residues seem to be mainly responsible for the differences in protein association in the crystal medium. These are Ser 103 and Leu 155 in gamma-II, which are replaced by Met 103 and His 155 in gamma-IIb. A similar substitution of these residues is observed in different gene products of gamma-crystallins from a number of vertebrates. This is consistent with the existence of a genetically controlled mechanism for determining intermolecular association of gamma-crystallins in the native medium of the lens.

Amino Acid Sequence↗
PubMed1988

Evolutionary and functional relationships between the basic and acidic beta-crystallins.

beta-Crystallins are complex oligomers composed of many related subunits. In order to understand their interactions we have built molecular models of several bovine beta-crystallins, based on their sequence similarity to the well-defined gamma-II crystallin structure, using interactive computer graphics techniques. Their common origin with gamma-crystallin is displayed in both the retention of four-fold sequence repeats of critical residues involved with stabilizing a folded beta-hairpin and the conservation of core-filling hydrophobic side-chains. The beta-crystallins have been built as bilobal molecules with each domain composed of two 'Greek key' motifs which associate about an approximate two-fold axis to form beta-sheets. The beta-crystallin sequences have previously been shown to comprise two families, the basic and acidic subunits, which have extensions of sequence. The three-dimensional models show how the two families appear to stabilize the folded beta-hairpin in the N- and C-terminal domains in ways which suggest that they have diverged from a common ancestor in different ways. Acidic beta-crystallins, like gamma-crystallins, have a regular array of charges on their N-terminal domain which has been interrupted in basic beta-crystallins by hydrophobic residues which may be related to the presence of a C-terminal extension. beta-Crystallins are more highly charged than gamma-crystallins although their charge density is higher in certain regions of the N-terminal domain, particularly in beta B1-crystallin. beta-crystallins also differ from gamma-crystallins in the virtual absence of core-filling sulphydryl groups whereas they have numerous sulphur-containing side-chains together with tryptophan and histidine rings protruding from the globular domains, particularly in the acidic subunits. The burial of these residues in subunit contacts is consistent with their spectroscopic and electrostatic properties. Protein subunit aggregation commonly occurs through hydrophobic interaction or beta-sheet extension. Analysis of the subunit surfaces has identified an N-terminal hydrophobic region common to beta B1 and beta B2 whereas a C-terminal hydrophobic loop region is common to beta B1 and beta A1 and may be correlated with their association properties. It is suggested that the polar C-terminal domain of beta B2 contributes towards the solubility of higher aggregates by interactions involving beta-sheet structure.

Amino Acid Sequence↗
PubMed1988

Duck lens epsilon-crystallin and lactate dehydrogenase B4 are identical: a single-copy gene product with two distinct functions.

To investigate whether or not duck lens epsilon-crystallin and duck heart lactate dehydrogenase (LDH) B4 are the product of the same gene, we have isolated and sequenced cDNA clones of duck epsilon-crystallin. By using these clones we demonstrate that there is a single-copy Ldh-B gene in duck and in chicken. In the duck lens this gene is overexpressed, and its product is subject to posttranslational modification. Reconstruction of the evolutionary history of the LDH protein family reveals that the mammalian Ldh-C gene most probably originated from an ancestral Ldh-A gene and that the amino acid replacement rate in LDH-C is approximately 4 times the rate in LDH-A. Molecular modeling of LDH-B sequences shows that the increased thermostability of the avian tetramer might be explained by mutations that increase the number of ion pairs. Furthermore, the replacement of bulky side chains by glycines on the corners of the duck protein suggests an adaptation to facilitate close packing in the lens.

Animals↗
PubMed1986

Structural variation in mammalian gamma-crystallins based on computer graphics analyses of human, rat and calf sequences. 1. Core packing and surface properties.

A comparison of mammalian gamma-crystallins has been made by computer-graphics model building of several gamma-crystallin sequences based on the atomic co-ordinates of the X-ray determined structure of calf gamma-II crystallin. The complete family of rat gamma-crystallins is compared together with the orthologous protein, gamma 1-2 crystallin, from rat, human and calf lens, and the orthologous protein, gamma 2-1 crystallin, from rat and human lens. In human gamma-crystallins, a major structural difference, the replacement of an arginine by a cysteine, occurs in one of the four-fold repeated folded hairpins, which may affect stability. Sequence variations involving buried residues were observed, leading to small differences in core packing of the different sequences which may be related to their regional location in the lens. Model-building studies also indicate that the surfaces of the different gamma-crystallins vary in number of exposed hydrophobic residues and ion pairs. These differences would affect protein-water interactions and therefore contribute to refractive index. A major variable region of the gamma-crystallin structures involves polar residues surrounding the inter-domain contact and the length of the polypeptide connecting the two domains. An attempt is made to correlate bovine gamma-crystallins which are known to be responsible for cold cataract with the corresponding sequences from rat lens.

Amino Acid Sequence↗
PubMed1985

The reaction of glutathione with the eye-lens protein gamma-crystallin.

Lens cells contain high concentrations of thiol-rich proteins, gamma-crystallins and reduced glutathione. Solutions of bovine gamma-crystallin react avidly with either reduced or oxidized glutathione to form protein-glutathione mixed disulphides. A method of purification of a gamma-II crystallin-glutathione adduct containing two mixed disulphide groups is described.

Animals↗