Biomedical subjects
Publications and source records attributed to C Slingsby.
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.
delta-Crystallin, purified from either adult turkey or quail lenses, and characterized by amino acid composition and circular dichroism spectroscopy, is shown to be similar to delta-crystallin purified from 3-day-old chick lenses. Crystallization of turkey delta-crystallin is reported along with the characterization of the crystal unit cell by X-ray diffraction techniques. Variation in unit cell parameters is observed and is correlated with hydration.
To determine the molecular mechanisms for cold cataract formation in the nucleus of the young mammalian lens, we have investigated the thermally reversible opacification of gamma-crystallin solutions isolated from calf lens. Coexistence curves (plots of opacification temperature Tc versus protein concentration) were determined for the individual gamma-crystallin fractions II, III, and IV as well as for the unfractionated gamma-crystallin mixtures isolated from the nucleus and cortex. The coexistence curve of gamma IV-crystallin is remarkably elevated above those of gamma II- and gamma III-crystallin and the gamma-crystallin mixtures. The gamma IV-crystallin fraction is the major determinant of the opacification temperature within the whole lens or isolated cytoplasm. Quasielastic light-scattering spectroscopy of gamma IV-crystallin solutions indicates that above Tc there are two populations of protein aggregates of distinctly different mean size. As the temperature is lowered towards Tc, both populations increase in size. Opacification occurs when the population of large scatterers, which is composed of less than 0.1% protein by weight, reaches an average radius of about 20,000 A.
Knowledge of the three-dimensional structure of bovine gamma II-crystallin has provided the basis for building molecular models using computer graphics of two human gamma-crystallins, the sequences of which have recently been determined. The tertiary structures of these gamma-crystallins are predicted to be highly conserved. They have extensive networks of interacting charges on their surfaces, which may contribute to their thermodynamic stability and partially define the degree of water retention in the lens. The human crystallins appear to be more hydrophobic than the bovine molecule. All have arrangements of cysteine thiols which may be important as electron sinks and reserve redox potential in the normal lens but which may contribute to protein aggregation in cataract.
The bovine beta-crystallin Bp chain is organized into two very similar domains, with short extensions at both N- and C-termini, and two alternative models for the beta Bp dimer have been proposed (Wistow, G., Slingsby, C., Blundell, T., Driessen, H.P.C., De Jong, W.W. and Bloemendal, H. (1981) FEBS Lett. 133, 9-16). By limited proteolysis the C-terminal arms can be cleaved off rapidly from the beta Bp dimer, while the N-terminal arms are more difficult to remove. Trypsin divides the beta Bp chain into two fragments which approximately correspond to the two structural domains. Dissociation and reassociation of the different products of limited proteolysis indicated that: the C-terminal arm extends freely from the surface and is not involved in subunit-contact; at least one N-terminal arm seems required for dimer formation; the N-terminal domains have a greater tendency to associate than the C-terminal domains and, when mixed, the purified domains reassociate partially to a Mr 50 000 structure like native beta Bp. These findings support the more extended dimer model of beta Bp.
We report the X-ray structure analysis and refinement at 1.9 A resolution of calf gamma-II crystallin, a lens-specific protein. The sequence of Croft (1972) has been modified to give a polypeptide chain of 174 residues (cf. 165). The protein has a symmetrical, hierarchical structure of two globular domains each comprising two similar "Greek key" motifs, consecutive along the polypeptide chain, and related by a pseudo 2-fold axis. The two domains pack together with a single connection and are related by a further pseudo 2-fold axis which bisects the angle between the intra-domain dyads. Forty-two pairs of C alpha positions for the two most similar motifs have root-mean-square separation at best fit of 0.69 A. The N and C-terminal domains gave root-mean-square separation of 0.89 A for 82 pairs of C alpha atoms at best fit. In each domain the two Greek key motifs form a pair of four-stranded antiparallel beta-pleated sheets, each sheet composed of three stands from one motif and one from the other. The sheets pack together in a wedge shape, closed at the top by the loops connecting the third and fourth strands of each motif. The first two strands of each motif form an extended beta-hairpin which is folded on to the beta-sheet. The packing of each motif into the globular domains involves a staggered bilayer of side-chains between each pair of beta-sheets which does not preserve the pseudo 2-fold axes observed in the C alpha position topology. In the core of each domain there are interactions between polarizable aromatic groups and sulphur-containing residues which may contribute to stability and may also serve to protect aromatic side-chains from ultraviolet light damage in the lens. At the surface of the molecule over half the ionic side-chains are closely paired, which probably stabilizes the tertiary fold and may reduce the water bound. Crystal lattice interactions are described which may be similar to those occurring in vivo in the lens between crystallins. Seven cysteine residues have been identified in the structure and these may have a role in the thermodynamic stability of the molecule, its intermolecular interactions under the normal reducing conditions of the lens, and also in the aggregation and cross-linking which occur in some forms of cataract. Three of these residues, Cys18, Cys23 and Cys74, form a cluster in the N-terminal domain.(ABSTRACT TRUNCATED AT 400 WORDS)
Monomeric crystallins, beta s and several members of the gamma-crystallin family, occur in different relative proportions in a way which is related to the age of the lens cells. Methods of purification of large amounts of these different low molecular weight protein components from young and old bovine lenses are described along with details of crystallization of several gamma-fractions. Purification procedures have been developed for rabbit gamma-crystallins. The chromatographic methods achieve separation of several bovine and rabbit gamma-crystallins which have very similar electrophoretic mobilities. However, on storage, many electrophoretic variants are generated from some of these fractions.
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Although lens structural proteins are extremely diverse, there appear to be common structural motifs or folding units from which the beta-gamma-crystallins can be constructed. X-ray diffraction techniques have solved the atomic three-dimensional structure of monomeric bovine gamma-II. The related protein, beta Bp, the principal subunit of bovine beta-crystallin has been predicted, using computer graphics techniques, to have a similar tertiary structure. These molecular models show the spatial orientation of some of the reactive side-chains which have been implicated in oxidative disruption of lens structure.
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The three-dimensional structure of the eye lens protein, bovine gamma-crystallin II, has been determined at 2.6 A resolution. The protein has a tow domain beta-structure, folded into four remarkably similar 'Greed key' motifs, and shows the highest internal symmetry of any protein studied by X-ray analysis. Although the symmetrical structure seems very stable, the arrangement of the sulphydryl groups would allow intramolecular cross-linking leading to possible destabilization, and intermolecular cross-linking leading to aggregation, both of which may be important to cataract formation.
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A 23,000 molecular weight beta-crystallin (beta 23) of the murine eye lens is encoded in a 4.1 +/- 0.3-kilobase gene containing three introns. Each of the four exons seems to code for a separate structural motif of the protein, whose tertiary structure was predicted by an interactive computer graphics technique based on the crystallographic structure of bovine gamma II-crystallin. The first exon also encodes a hydrophobic N-terminal peptide resembling membrane anchor sequences of other proteins. Our results indicate structural homology among the beta- and gamma-crystallin polypeptides, and link gene structure with protein structure in this superfamily of lens proteins.
A comparative study of intramolecular crystal interactions of two homologous gamma-crystallins II and IIIb from calf lens has been carried out. It has been shown that the key role in formation of "dimeric" associates of the head-to-tail type for gamma-crystallin IIIb is played by Met-103 which is located in the middle of the hydrophobic surface region. The absence of such a region in the molecule of gamma-crystallin II is explained by replacement of Met-103 by Ser-103. A similar alternative with the exchange of the hydrophobic residue by the hydrophilic one is observed for different gene products of gamma-crystallins from a number of vertebrates. This suggests intermolecular interaction of gamma-crystallins in the native medium of the lens.