Structural studies on bovine -crystallin.
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ABSTRACT:
SUBMITTER: Croft LR
PROVIDER: S-EPMC1176593 | biostudies-other | 1971 Feb
REPOSITORIES: biostudies-other
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Project description:1. alpha-Crystallin has been isolated from the cortex of ox lens by isoelectric precipitation followed by chromatography on DEAE-cellulose. The amino acid composition is in agreement with that reported for alpha-crystallin prepared by a different method. There is one thiol group/20000g. of protein (20000 is the order of magnitude of the sub-unit molecular weight), and disulphide bonds are absent. 2. The thiol group has been alkylated with radioactive iodoacetate in the presence of urea. 3. Partial acid hydrolysis of the alkylated protein gives, according to the conditions, mainly three radioactive peptides or nearly exclusively one radioactive dipeptide. The dipeptide is N-seryl-(S-carboxymethyl)cysteine, Ser-CMCys. The two other peptides are probably the tripeptides related to Ser-CMCys. 4. The simplest interpretation of these results is that the sequence around the cysteine residue is a common structural feature of the sub-units of alpha-crystallin.
| S-EPMC1207209 | biostudies-other
Project description:Our previous work has shown that the treatment of bovine rhodopsin with the proteolytic enzyme papain gives rise to a cleaved, but fully functional, complex consisting of three fragments, H, M and L (heavy, medium and light), held together by strong non-covalent forces. By using some of the chemical and physical differences between the three fragments, a protocol for the preparative isolation of each fragment was devised. Purified M-fragment, which had been radiochemically labelled at the retinal-binding site was treated with CNBr and the mixture subjected to a multi-step separation to furnish a retinyl peptide. The sequence analysis of the latter showed that the retinal-binding lysine residue was located at position 296 from the N-terminal of rhodopsin (or residue 53 from the C-terminal). In order to ascertain the position of the cytoplasmic loop which exists between the M- and L-fragments, radiochemically labelled L-fragment was isolated from the cleaved complex. The purified L-fragment was shown to consist of two populations of peptides which were produced by the action of papain on the bonds between Lys-311 and Gln-312 and between Gln-312 and Phe-313.
| S-EPMC1154327 | biostudies-other
Project description:The single-banding components of bovine transferrin from animals homozygous for the four transferrin variants found in the U.K. were isolated. Sedimentation equilibrium ultracentrifugation and sodium dodecyl sulphate-polyacrylamide-gel electrophoresis showed that the bands of a single variant have molecular weights of 77500 and 73300 respectively. The different bands of a single variant and single bands of different variants show no evidence of size heterogeneity or of low-molecular-weight peptides being split off after reduction in 6m-guanidine hydrochloride. The two slower bands of a single variant, which both contain 2 molecules of sialic acid/molecule of protein, have the same molecular weight and amino acid composition, and give identical peptide ;maps', although differences in composition and peptide ;maps' occur between the different variants. The results support the concept that bovine transferrin is essentially a single polypeptide chain, but they do not explain differences in electrophoretic mobility between bands of the same variant which are not produced by differing sialic acid content.
| S-EPMC1165792 | biostudies-other
Project description:?-Crystallin is the major protein of the eye lens and a member of the family of small heat-shock proteins. Its concentration in the human eye lens is extremely high (about 450 mg/mL). Three-dimensional structure of native ?-crystallin is unknown. First of all, this is the result of the highly heterogeneous nature of ?-crystallin, which hampers obtaining it in a crystalline form. The modeling based on the electron microscopy (EM) analysis of ?-crystallin preparations shows that the main population of the ?-crystallin polydisperse complex is represented by oligomeric particles of rounded, slightly ellipsoidal shape with the diameter of about 13.5 nm. These complexes have molecular mass of about 700 kDa. In our opinion, the heterogeneity of the ?-crystallin complex makes it impossible to obtain a reliable 3D model. In the literature, there is evidence of an enhanced chaperone function of ?-crystallin during its dissociation into smaller components. This may indirectly indicate that the formation of heterogeneous complexes is probably necessary to preserve ?-crystallin in a state inactive before stressful conditions. Then, not only the heterogeneity of the ?-crystallin complex is an evolutionary adaptation that protects ?-crystallin from crystallization but also the enhancement of the function of ?-crystallin during its dissociation is also an evolutionary acquisition. An analysis of the literature on the study of ?-crystallin in vitro led us to the assumption that, of the two ?-crystallin isoforms (?A- and ?B-crystallins), it is ?A-crystallin that plays the role of a special chaperone for ?B-crystallin. In addition, our data on X-ray diffraction analysis of ?-crystallin at the sample concentration of about 170-190 mg/mL allowed us to assume that, at a high concentration, the eye lens ?-crystallin can be in a gel-like stage. Finally, we conclude that, since all the accumulated data on structural-functional studies of ?-crystallin were carried out under conditions far from native, they cannot adequately reflect the features of the functioning of ?-crystallin in vivo.
| S-EPMC7235859 | biostudies-literature
Project description:The small heat shock protein alphaB-crystallin (alphaB) contributes to cellular protection against stress. For decades, high-resolution structural studies on oligomeric alphaB have been confounded by its polydisperse nature. Here, we present a structural basis of oligomer assembly and activation of the chaperone using solid-state NMR and small-angle X-ray scattering (SAXS). The basic building block is a curved dimer, with an angle of approximately 121 degrees between the planes of the beta-sandwich formed by alpha-crystallin domains. The highly conserved IXI motif covers a substrate binding site at pH 7.5. We observe a pH-dependent modulation of the interaction of the IXI motif with beta4 and beta8, consistent with a pH-dependent regulation of the chaperone function. N-terminal region residues Ser59-Trp60-Phe61 are involved in intermolecular interaction with beta3. Intermolecular restraints from NMR and volumetric restraints from SAXS were combined to calculate a model of a 24-subunit alphaB oligomer with tetrahedral symmetry.
| S-EPMC2957905 | biostudies-literature
Project description:The depolymerization of bovine cervical glycoprotein resulting from cleavage of disulphide bonds. Pronase digestion and both procedures sequentially was assessed by using gel filtration. Cleavage of disulphide bonds followed by Pronase digestion produced more extensive depolymerization than did either treatment alone, and gel filtration of the products resulted in two major peaks of glycosylated material on Sepharose CL-2B and Sepharose 4B. The glycopolypeptides in both peaks had similar sugar and sulphate compositions, but they migrated to different extents on gel electrophoresis. Electrophoretic studies indicated that both glycopolypeptides were derived from the same glycoprotein molecule and not from a mixture of two similar glycoproteins. Pronase digestion of glycoproteins in which the disulphide bonds had been labelled with iodo-[1-14C]acetamide revealed that most of the cysteine residues were situated in regions susceptible to Pronase. The results show the presence of two types of structural regions in bovine cervical glycoprotein, namely 'naked' peptide or non-glycosylated regions and glycopolypeptide subunit regions in which glycopolypeptides of two different sizes predominate. Comparison of the cervical glycoproteins isolated from mucus secreted during oestrus and pregnancy, by the methods outlined above, did not reveal any structural differences in the glycoproteins to explain the different physical properties of the mucus secreted under these conditions.
| S-EPMC1185863 | biostudies-other
Project description:αB-crystallin (heat shock protein β5/HSPB5) is a member of the family of small heat shock proteins that is expressed in various organs of the human body including eye lenses and muscles. Therefore, mutations in the gene of this protein (CRYAB) might have many pathological consequences. A new mutation has recently been discovered in the α-crystallin domain of this chaperone protein which replaces aspartate 109 with alanine (D109A). This mutation can cause myofibrillar myopathy (MFM), cataracts, and cardiomyopathy. In the current study, several spectroscopic and microscopic analyses, as well as gel electrophoresis assessment were applied to elucidate the pathogenic contribution of human αB-crystallin bearing D109A mutation in development of eye lens cataract and myopathies. The protein oligomerization, chaperone-like activity and chemical/thermal stabilities of the mutant and wild-type protein were also investigated in the comparative assessments. Our results suggested that the D109A mutation has a significant impact on the important features of human αB-crystallin, including its structure, size of the protein oligomers, tendency to form amyloid fibrils, stability, and chaperone-like activity. Given the importance of aspartate 109 in maintaining the proper structure of the α-crystallin domain, its role in the dimerization and chaperone-like activity, as well as preserving protein stability through the formation of salt bridges; mutation at this important site might have critical consequences and can explain the genesis of myopathy and cataract disorders. Also, the formation of large light-scattering aggregates and disruption of the chaperone-like activity by D109A mutation might be considered as important contributing factors in development of the eye lens opacity.
| S-EPMC8629256 | biostudies-literature
Project description:Delta crystallin, a taxon-specific crystallin present in avian eye lenses, is homologous to the urea cycle enzyme ASL (argininosuccinate lyase). Although there are two delta crystallin isoforms in duck lenses, ddeltac1 (duck delta1 crystallin) and ddeltac2 (duck delta2 crystallin), only ddeltac2 is catalytically active. Previous structural studies have suggested that residues Ser283 and His162 in the multi-subunit active site of ddeltac2/ASL are the putative catalytic acid/base, while the highly conserved, positively charged Lys289 is thought to help stabilize the carbanion intermediate. The strict conservation of a small hydroxy-containing residue (Thr or Ser) at position 161 adjacent to the putative catalytic base, as well as its proximity to the substrate in the S283A ddeltac2 enzyme-substrate complex, prompted us to investigate further the role this residue. Structures of the active T161S and inactive T161D ddeltac2 mutants, as well as T161D complexed with argininosuccinate, have been determined to 2.0 A resolution. The structures suggest that a hydroxy group is required at position 161 to help correctly position the side chain of Lys289 and the fumarate moiety of the substrate. Threonine is probably favoured over serine, because the interaction of its methyl group with Leu206 would restrict its conformational flexibility. Residues larger than Thr or Ser interfere with substrate binding, supporting previous suggestions that correct positioning of the substrate's fumarate moiety is essential for catalysis to occur. The presence of the 280s loop (i.e. a loop formed by residues 270-290) in the 'open' conformation suggests that loop closure, thought to be essential for sequestration of the substrate, may be triggered by the formation of the carbanion or aci-carboxylate intermediates, whose charge distribution more closely mimics that of the sulphate ion found in the active-site region of the inactive ddeltac1. The 280s loop in ddeltac1 is in the closed conformation.
| S-EPMC1134128 | biostudies-literature
Project description:We model screened, site-specific charge regulation of the eye lens protein bovine gammaB-crystallin (?B) and study the probability distributions of its proton occupancy patterns. Using a simplified dielectric model, we solve the linearized Poisson-Boltzmann equation to calculate a 54×54 work-of-charging matrix, each entry being the modeled voltage at a given titratable site, due to an elementary charge at another site. The matrix quantifies interactions within patches of sites, including ?B charge pairs. We model intrinsic pK values that would occur hypothetically in the absence of other charges, with use of experimental data on the dependence of pK values on aqueous solution conditions, the dielectric model, and literature values. We use Monte Carlo simulations to calculate a model grand-canonical partition function that incorporates both the work-of-charging and the intrinsic pK values for isolated ?B molecules and we calculate the probabilities of leading proton occupancy configurations, for 4<pH<8 and Debye screening lengths from 6 to 20 Å. We select the interior dielectric value to model ?B titration data. At pH 7.1 and Debye length 6.0 Å, on a given ?B molecule the predicted top occupancy pattern is present nearly 20% of the time, and 90% of the time one or another of the first 100 patterns will be present. Many of these occupancy patterns differ in net charge sign as well as in surface voltage profile. We illustrate how charge pattern probabilities deviate from the multinomial distribution that would result from use of effective pK values alone and estimate the extents to which ?B charge pattern distributions broaden at lower pH and narrow as ionic strength is lowered. These results suggest that for accurate modeling of orientation-dependent ?B-?B interactions, consideration of numerous pairs of proton occupancy patterns will be needed.
| S-EPMC5830141 | biostudies-literature
Project description:Two populations of alkaline-borohydride-reduced keratan sulphate (KS) chains were prepared from the two peptido-keratan sulphate trypsin fragments of proteoglycan aggregates isolated from bovine femoral head cartilage (6-year-old animals). Each population was separated by high-performance ion-exchange chromatography on a Pharmacia Mono-Q column into eight pools, Q1-Q8. These were analysed by gel permeation chromatography, radioimmunoassay with the monoclonal antibody MZ15, and 500 MHz 1H n.m.r. spectroscopy. Upon chromatography on Sephadex G-75 the Mono-Q fractions were shown to increase in hydrodynamic size progressively from Q1 to Q8 for both KS populations. For each population the strongest antigenic response with the anti-KS monoclonal antibody MZ15 was expressed by the two fractions of greatest size and charge density, Q7 and Q8. Proton n.m.r. spectroscopic studies on the two series of fractions demonstrated: (i) a progressive increase in the level of galactose sulphation from Q1 to Q8, (ii) the presence of approximately one alpha(1-3)-linked fucose residue per chain in every sample, and (iii) the presence of N-acetylneuraminic acids in three discrete environments, two alpha(2-3)- and one alpha(2-6)-linked in every sample. The results are discussed in terms of a possible heterogeneity in the carbohydrate-protein linkage region of keratan sulphates from bovine articular cartilage.
| S-EPMC1138657 | biostudies-other