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Anushree Roy - One of the best experts on this subject based on the ideXlab platform.
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Distribution of protein Ramachandran psi (ψ) angle using non-resonance visible raman scattering measurements.
The journal of physical chemistry. B, 2013Co-Authors: Susmita Bhattacharya, Sudeshna Ghosh, Nitin Pandey, Susmitnarayan Chaudhury, Swagata Dasgupta, Anushree RoyAbstract:Knowing the distribution of Ramachandran angles helps in understanding peptide and protein backbone conformation. Empirical relations are proposed to correlate the spectral profile of the amide III3 band, obtained from ultraviolet resonance Raman measurements (UVRR), with the Ramachandran dihedral psi angle distribution in small peptide and protein molecules, in different environmental conditions (Mikhonin et al. J. Phys. Chem. B 2006, 110, 1928–1943). It has also been used for more complicated structures, like large globular proteins and protein fibrils. In our work here, we use visible Raman spectra and available empirical relations to obtain similar correlations for human serum albumin, hen egg white lysozyme, and human Gamma Crystallin. We also report the dihedral angle distribution in fibrils and a denatured protein in an ethanol environment using the same spectroscopic technique.
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Distribution of Protein Ramachandran Psi (ψ) Angle Using Non-Resonance Visible Raman Scattering Measurements B
The Journal of Physical Chemistry, 2013Co-Authors: Susmita Bhattacharya, Sudeshna Ghosh, Nitin Pandey, Susmitnarayan Chaudhury, Swagata Dasgupta, Anushree RoyAbstract:Knowing the distribution of Ramachandran angles helps in understanding peptide and protein backbone conformation. Empirical relations are proposed to correlate the spectral profile of the amide III₃ band, obtained from ultraviolet resonance Raman measurements (UVRR), with the Ramachandran dihedral psi angle distribution in small peptide and protein molecules, in different environmental conditions (Mikhonin et al. J. Phys. Chem. B 2006, 110, 1928–1943). It has also been used for more complicated structures, like large globular proteins and protein fibrils. In our work here, we use visible Raman spectra and available empirical relations to obtain similar correlations for human serum albumin, hen egg white lysozyme, and human Gamma Crystallin. We also report the dihedral angle distribution in fibrils and a denatured protein in an ethanol environment using the same spectroscopic technique.
Abraham Spector - One of the best experts on this subject based on the ideXlab platform.
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alpha Crystallin can act as a chaperone under conditions of oxidative stress
Investigative Ophthalmology & Visual Science, 1995Co-Authors: Keyang Wang, Abraham SpectorAbstract:PURPOSE: Previous studies have shown that alpha-Crystallin, a major lens protein, acts as a chaperone preventing the thermal denaturation of other lens Crystallins. However, there has not been an examination of the alpha-Crystallin chaperone ability with respect to the types of insult thought to cause human cataract. Therefore, an examination of the chaperone potential of alpha-Crystallin under conditions of oxidative stress was undertaken. METHODS: Oxidation of alpha-, beta low (beta L)-, and Gamma-Crystallins was performed with an ascorbate FeCl3-EDTA-H2O2 system. Thermal denaturation was carried out by heating preparations at 62 degrees C or 72 degrees C. After protein denaturation, 360 nm scatter was measured. Protein-complex formation was measured with a TSK gel G4000 SW 600 x 7.5 mm exclusion column. RESULTS: This study indicates that: (1) alpha-Crystallin markedly reduces the 360-nm light scatter of Gamma-Crystallin caused by oxidation at 37 degrees C. (2) alpha-Crystallin appears to protect the Gamma-Crystallin thiol groups from extensive oxidation. (3) Oxidation of alpha-Crystallin causes only a small change in its ability to prevent heat-induced scattering of either Gamma- or beta L-Crystallin. (4) Oxidation of both alpha- and Gamma-Crystallin does not significantly affect the ability of alpha-Crystallin to inhibit 360-nm light scattering of Gamma-Crystallin at 72 degrees C. (5) Oxidation of beta L-Crystallin decreases its susceptibility to thermally induced scattering, but, conversely, oxidation of Gamma-Crystallin increases such susceptibility. (6) Oxidation of beta L-Crystallin at 37 degrees C produces only a slight increase in light scatter, in contrast to observations obtained with Gamma-Crystallin. (7) alpha-Crystallin provides long-term protection against thermally induced scatter of beta L-Crystallin but not of Gamma-Crystallin. High-performance liquid chromatography (HPLC) analysis suggests that the alpha-Gamma-Crystallin complex gradually becomes insoluble at 72 degrees C, in contrast to the alpha-beta L-Crystallin complex. Differing from thermal insult, alpha-Crystallin causes a marked decrease in Gamma-Crystallin light scattering under long-term oxidation. (8) The alpha-Gamma-Crystallin complex that results from oxidation represents a weak interaction because it cannot be isolated with procedures used to obtain the thermally induced complex. (9) This work confirms a previous study demonstrating that each alpha monomer (alpha m) contains a binding site for a partially denatured Crystallin. CONCLUSIONS: The overall results indicate that alpha-Crystallin can act as a chaperone under conditions of oxidative stress, decreasing the light scatter and thiol oxidation of other Crystallins. Because oxidative stress is thought to be present under normal physiological conditions, it is probable that alpha-Crystallin contributes to the mechanisms that maintain the lens in a transparent state.
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alpha Crystallin can act as a chaperone under conditions of oxidative stress
Investigative Ophthalmology & Visual Science, 1995Co-Authors: Keyang Wang, Abraham SpectorAbstract:PURPOSE: Previous studies have shown that alpha-Crystallin, a major lens protein, acts as a chaperone preventing the thermal denaturation of other lens Crystallins. However, there has not been an examination of the alpha-Crystallin chaperone ability with respect to the types of insult thought to cause human cataract. Therefore, an examination of the chaperone potential of alpha-Crystallin under conditions of oxidative stress was undertaken. METHODS: Oxidation of alpha-, beta low (beta L)-, and Gamma-Crystallins was performed with an ascorbate FeCl3-EDTA-H2O2 system. Thermal denaturation was carried out by heating preparations at 62 degrees C or 72 degrees C. After protein denaturation, 360 nm scatter was measured. Protein-complex formation was measured with a TSK gel G4000 SW 600 x 7.5 mm exclusion column. RESULTS: This study indicates that: (1) alpha-Crystallin markedly reduces the 360-nm light scatter of Gamma-Crystallin caused by oxidation at 37 degrees C. (2) alpha-Crystallin appears to protect the Gamma-Crystallin thiol groups from extensive oxidation. (3) Oxidation of alpha-Crystallin causes only a small change in its ability to prevent heat-induced scattering of either Gamma- or beta L-Crystallin. (4) Oxidation of both alpha- and Gamma-Crystallin does not significantly affect the ability of alpha-Crystallin to inhibit 360-nm light scattering of Gamma-Crystallin at 72 degrees C. (5) Oxidation of beta L-Crystallin decreases its susceptibility to thermally induced scattering, but, conversely, oxidation of Gamma-Crystallin increases such susceptibility. (6) Oxidation of beta L-Crystallin at 37 degrees C produces only a slight increase in light scatter, in contrast to observations obtained with Gamma-Crystallin. (7) alpha-Crystallin provides long-term protection against thermally induced scatter of beta L-Crystallin but not of Gamma-Crystallin. High-performance liquid chromatography (HPLC) analysis suggests that the alpha-Gamma-Crystallin complex gradually becomes insoluble at 72 degrees C, in contrast to the alpha-beta L-Crystallin complex. Differing from thermal insult, alpha-Crystallin causes a marked decrease in Gamma-Crystallin light scattering under long-term oxidation. (8) The alpha-Gamma-Crystallin complex that results from oxidation represents a weak interaction because it cannot be isolated with procedures used to obtain the thermally induced complex. (9) This work confirms a previous study demonstrating that each alpha monomer (alpha m) contains a binding site for a partially denatured Crystallin. CONCLUSIONS: The overall results indicate that alpha-Crystallin can act as a chaperone under conditions of oxidative stress, decreasing the light scatter and thiol oxidation of other Crystallins. Because oxidative stress is thought to be present under normal physiological conditions, it is probable that alpha-Crystallin contributes to the mechanisms that maintain the lens in a transparent state.
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The calf Gamma Crystallins--a Raman spectroscopic study.
Experimental eye research, 1991Co-Authors: Jayanti Pande, Martin J. Mcdermott, Robert Callender, Abraham SpectorAbstract:The solution structures of the four major components of bovine lens Gamma-Crystallin, Gamma s, Gamma II, Gamma III and Gamma IV are compared using Raman spectroscopy. The spectral region sensitive to the vibrational frequencies of aromatic and sulfur containing residues and to the backbone skeletal stretching modes (500-1000 cm-1), and that reflecting secondary structure (1,000-1,700 cm-1) are strikingly similar in all four Gamma-Crystallin fractions. These similarities are indicative of the dominant anti-parallel beta sheet structure common to all the Gamma-Crystallins. A comparison of the ratios of the Raman intensities at 850 cm-1 and 830 cm-1 (I850/I830), an empirical measure of the degree of hydrogen bonding of phenolic hydroxyl groups, suggests that the tyrosine residues in all the Gamma-Crystallin fractions are moderately hydrogen bonded. Distinct differences in the solution structures of the Gamma-Crystallins were observed in the higher energy end of the vibrational Raman spectra. The sulfhydryl stretching frequencies for the Gamma-Crystallins exhibit complex splitting patterns in the 2,500-2,600 cm-1 region. These patterns are due to the competing effects of hydrogen bonding and S-pi interactions with neighboring aromatic residues. All five proteins exhibit multiple, but distinct, thiol frequencies, suggesting that the microenvironments of the cysteine residues in these proteins are significantly different.
Larry J. Takemoto - One of the best experts on this subject based on the ideXlab platform.
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Screening of Crystallin-Crystallin interactions using microequilibrium dialysis.
Molecular vision, 2005Co-Authors: Aldo Ponce, Larry J. TakemotoAbstract:PURPOSE It has been hypothesized that short-range, protein-protein interactions of Crystallin are necessary for the maintenance of lens transparency. Because of their probable weak nature, it has been difficult to both detect and quantitate the nature of these interactions. To determine if interactions exist between alpha-Crystallin and Gamma-Crystallin under true equilibrium conditions, we have used microequilibrium dialysis. METHODS Total alpha-Crystallin and Gamma-Crystallin were prepared from soluble proteins of fetal bovine lenses by HPLC and gel filtration chromatography. The proteins were added to one side of a microequilibrium dialysis cell, comprised of two chambers separated by a membrane with 100 kDa molecular weight cut-off. After reaching equilibrium, the amount of free Gamma-Crystallin and the amount of Gamma-Crystallin bound to alpha-Crystallin was determined by HPLC and reverse phase analysis of both chambers. Selected Gamma-Crystallin that bound to alpha-Crystallin was further purified by ion exchange chromatography, and then incubated with alpha-Crystallin, to verify the specificity of their binding. RESULTS Analysis of both microequilibrium dialysis chambers incubated at different times at 37 degrees C indicated that equilibrium was reached at 4 days. When total alpha-Crystallin and Gamma-Crystallin were incubated for this time period, significant binding was observed between alpha-Crystallin and the IIIA, II, and IVA species of Gamma-Crystallin. These interactions were confirmed by microequilibrium dialysis determinations containing alpha-Crystallin and purified Gamma-Crystallin species. CONCLUSIONS These results show that microequilibrium dialysis can be used to demonstrate significant noncovalent interactions of alpha-Crystallin and Gamma-Crystallin under true equilibrium conditions.
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Preferential interaction of alpha Crystallin with denatured forms of Gamma Crystallin
Investigative Ophthalmology & Visual Science, 1994Co-Authors: Daniel L. Boyle, Larry J. TakemotoAbstract:Purpose. To characterize the possible interaction of alpha Crystallin with partially denatured forms of Gamma Crystallin. Methods. Gamma Crystallin was denaturated in the presence of guanidine hydrochloride, then dialyzed in the presence or absence of alpha Crystallin. The high-molecular-weight complex formed in the presence of alpha was characterized by gel filtration chromatography, electron microscopy, and quantitative Western blot analysis. Results. Relative to native alpha or reconstituted aggregated of purified alpha, the higher molecular weight complex possessed a greater mean diameter and contained increased amounts of Gamma Crystallin
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Preferential interaction of alpha Crystallin with denatured forms of Gamma Crystallin.
Investigative ophthalmology & visual science, 1994Co-Authors: S. Gopalakrishnan, Daniel L. Boyle, Larry J. TakemotoAbstract:PURPOSE To characterize the possible interaction of alpha Crystallin with partially denatured forms of Gamma Crystallin. METHODS Gamma Crystallin was denatured in the presence of guanidine hydrochloride, then dialyzed in the presence or absence of alpha Crystallin. The high-molecular-weight complex formed in the presence of alpha was characterized by gel filtration chromatography, electron microscopy, and quantitative Western blot analysis. RESULTS Relative to native alpha or reconstituted aggregates of purified alpha, the higher molecular weight complex possessed a greater mean diameter and contained increased amounts of Gamma Crystallin. CONCLUSIONS Alpha Crystallin preferentially interacts with partially denatured forms of a lens protein, consistent with its putative role as a functional molecular chaperone in the intact lens.
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Binding of actin to lens alpha Crystallins
Current Eye Research, 1992Co-Authors: S. Gopalakrishnan, Larry J. TakemotoAbstract:Actin has been coupled to a cyanogen bromide - activated Sepharose 4B column, then tested for binding to alpha, beta, and Gamma Crystallin preparations from the bovine lens. Alpha, but not beta or Gamma, Crystallins bound to the actin affinity column in a time dependent and saturable manner. Subfractionation of the alpha Crystallin preparation into the alpha-A and alpha-B species, followed by incubation with the affinity column, demonstrated that both species bound approximately the same. Together, these studies demonstrate a specific and saturable binding of lens alpha-A and alpha-B with actin.
Susmita Bhattacharya - One of the best experts on this subject based on the ideXlab platform.
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Distribution of protein Ramachandran psi (ψ) angle using non-resonance visible raman scattering measurements.
The journal of physical chemistry. B, 2013Co-Authors: Susmita Bhattacharya, Sudeshna Ghosh, Nitin Pandey, Susmitnarayan Chaudhury, Swagata Dasgupta, Anushree RoyAbstract:Knowing the distribution of Ramachandran angles helps in understanding peptide and protein backbone conformation. Empirical relations are proposed to correlate the spectral profile of the amide III3 band, obtained from ultraviolet resonance Raman measurements (UVRR), with the Ramachandran dihedral psi angle distribution in small peptide and protein molecules, in different environmental conditions (Mikhonin et al. J. Phys. Chem. B 2006, 110, 1928–1943). It has also been used for more complicated structures, like large globular proteins and protein fibrils. In our work here, we use visible Raman spectra and available empirical relations to obtain similar correlations for human serum albumin, hen egg white lysozyme, and human Gamma Crystallin. We also report the dihedral angle distribution in fibrils and a denatured protein in an ethanol environment using the same spectroscopic technique.
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Distribution of Protein Ramachandran Psi (ψ) Angle Using Non-Resonance Visible Raman Scattering Measurements B
The Journal of Physical Chemistry, 2013Co-Authors: Susmita Bhattacharya, Sudeshna Ghosh, Nitin Pandey, Susmitnarayan Chaudhury, Swagata Dasgupta, Anushree RoyAbstract:Knowing the distribution of Ramachandran angles helps in understanding peptide and protein backbone conformation. Empirical relations are proposed to correlate the spectral profile of the amide III₃ band, obtained from ultraviolet resonance Raman measurements (UVRR), with the Ramachandran dihedral psi angle distribution in small peptide and protein molecules, in different environmental conditions (Mikhonin et al. J. Phys. Chem. B 2006, 110, 1928–1943). It has also been used for more complicated structures, like large globular proteins and protein fibrils. In our work here, we use visible Raman spectra and available empirical relations to obtain similar correlations for human serum albumin, hen egg white lysozyme, and human Gamma Crystallin. We also report the dihedral angle distribution in fibrils and a denatured protein in an ethanol environment using the same spectroscopic technique.
Rainer Jaenicke - One of the best experts on this subject based on the ideXlab platform.
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Homo-dimeric spherulin 3a: a single-domain member of the beta Gamma-Crystallin superfamily.
Biological chemistry, 1999Co-Authors: Michael Kretschmar, E.-m. Mayr, Rainer JaenickeAbstract:The beta Gamma-Crystallin superfamily of eye lens proteins comprises a class of structurally related members with a wide variety of different functions. Common features of these proteins are 1. the Greek-key motif of antiparallel beta-sheets, called the Crystallin fold, and 2. the high intrinsic long-term stability. Spherulin 3a (S3a), a dormant protein from the spherules of Physarum polycephalum, is the only known single-domain protein within the beta Gamma-Crystallin family. Based on sequence homology and 'domain swapping', it has been proposed to represent an evolutionary ancestor of present-day eye lens Crystallins. Since S3a is highly expressed in spherulating plasmodia of P. polycephalum under a variety of stress conditions, it can be assumed that the protein may serve as a compatible solute in the cytosol of the slime mold. In order to investigate the stability and other physicochemical properties of a single-domain all-beta protein, we isolated natural S3a. For the large-scale purification, the recombinant protein was cloned and expressed in Escherichia coli. The detailed spectral and biochemical analysis proved the recombinant protein to be authentic. In its native form, S3a is dimeric. Due to its exposed cysteine residues (Cys4), in the absence of reducing agents intermolecular disulfide cross-linking leads to the formation of higher oligomers. In order to preserve the native quaternary structure without aggregation artifacts in denaturation/renaturation experiments, the Cys4-->Ser mutant (S3a C4S) was produced. Both the wild-type protein and its mutant are indistinguishable in their physicochemical properties. At pH 3 - 4, both proteins form a stable compact intermediate (A-state). Concentration-dependent thermal and chemical denaturation showed that the equilibrium unfolding of S3a obeys the simple two-state model with no significant occurrence of folding intermediates.
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The C-terminal domains of GammaS-Crystallin pair about a distorted twofold axis.
Protein engineering, 1998Co-Authors: Ajit K. Basak, Rainer Jaenicke, N. H. Lubsen, R. C. Kroone, C.e. Naylor, C SlingsbyAbstract:The 2-domain GammaS-Crystallin, a highly conserved early evolutionary off-shoot of the Gamma-Crystallin family, is located in the water-rich region of eye lenses. The expressed C-terminal domain, GammaS-C, has been crystallized and the 2.56 A X-ray structure determined. There are two domains in the asymmetric unit which pair about a distorted twofold axis. One of the domains has an altered conformation in a highly conserved region of the protein, the tyrosine corner. The distorted GammaS-C dimer of domains is compared with the highly symmetrical, equivalent recombinant dimer of C-terminal domains from GammaB-Crystallin. Sequence changes close to the interface, that distinguish GammaS from the other Gamma-Crystallins, are examined in order to evaluate their role in symmetrical domain pairing.
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Eye-lens proteins: structure, superstructure, stability, genetics.
Naturwissenschaften, 1994Co-Authors: Rainer JaenickeAbstract:: The eye lens in vertebrates and invertebrates is an avascular tissue which allows one to focus objects on the retina. The lens grows throughout life, maintaining transparency without significant turnover of its densely packed proteins. Apart from cytoskeletal and taxon-specific components, these proteins belong mainly to the alpha- and beta Gamma-Crystallin families. The detailed structural analysis of beta Gamma-Crystallins can explain the anomalous stability by the specific supersecondary structure ("Greek key" topology) of the domains and by strong domain and subunit interactions. The spatial correlation of the molecules at the given high concentrations in the fiber cells gives rise to "short-range order" with minimum light scattering, thus providing optimum transparency of the eye lens.
