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Y V Sergeev

Publications and source records attributed to Y V Sergeev.

10 recordsLinked to original sources
PubMed2004

Energetics of domain-domain interactions and entropy driven association of beta-crystallins.

Beta-crystallins are major protein constituents of the mammalian lens, where their stability and association into higher order complexes are critical for lens clarity and refraction. They undergo modification as the lens ages, including cleavage of their terminal extensions. The energetics of betaA3- and betaB2-crystallin association was studied using site-directed mutagenesis and analytical ultracentrifugation. Recombinant (r) murine wild type betaA3- and betaB2-crystallins were modified by removal of either the N-terminal extension of betaA3 (rbetaA3Ntr) or betaB2 (rbetaB2Ntr), or both the N- and C-terminal extensions of betaB2 (rbetaB2NCtr). The proteins were expressed in Sf9 insect cells or Escherichia coli and purified by gel-filtration and ion-exchange chromatography. All beta-crystallins studied demonstrated fast reversible monomer-dimer equilibria over the temperature range studied (5-35 degrees C) with a tendency to form tighter dimers at higher temperatures. The N-terminal deletion of rbetaA3 (rbetaA3Ntr) significantly increases the enthalpy (+10.9 kcal/mol) and entropy (+40.7 cal/deg mol) of binding relative to unmodified protein. Removal of both N- and C-terminal extensions of rbetaB2 also increases these parameters but to a lesser degree. Deletion of the betaB2-crystallin N-terminal extension alone (rbetaB2Ntr) gave almost no change relative to rbetaB2. The resultant net negative changes in the binding energy suggest that betaAlpha3- and betaB2-crystallin association is entropically driven. The thermodynamic consequences of the loss of betaAlpha3-crystallin terminal extensions by in vivo proteolytic processing could increase their tendency to associate and so promote the formation of higher order associates in the aging and cataractous lens.

Amino Acid Sequence↗
PubMed2004

Beta-crystallin association.

Beta-crystallins are major protein constituents of the mammalian lens, where their stability and association into higher order complexes are critical for lens clarity and refraction. Dimerization is an initial step in formation of beta-crystallin complexes. Beta-crystallin association into dimers is energetically highly favoured, but rapidly reversible under physiological conditions. Beta-crystallin dimers can exchange monomers, probably through a transient and energetically unfavoured monomer intermediate state. As predicted by molecular modelling, the fraction of beta-crystallin present as dimers increases with increasing temperature, implying that beta-crystallin association is entropically driven.

Chromatography, Gel↗
PubMed2004

Beta-crystallin association.

Beta-crystallins are major protein constituents of the mammalian lens, where their stability and association into higher order complexes are critical for lens clarity and refraction. Dimerization is an initial step in formation of beta-crystallin complexes. Beta-crystallin association into dimers is energetically highly favoured, but rapidly reversible under physiological conditions. Beta-crystallin dimers can exchange monomers, probably through a transient and energetically unfavoured monomer intermediate state. As predicted by molecular modelling, the fraction of beta-Crystallin present as dimers increases with increasing temperature, implying that beta-crystallin association is entropically driven.

Chromatography, Gel↗
PubMed2002

Comparison of ultraviolet induced photo-kinetics for lens-derived and recombinant beta-crystallins.

PURPOSE: The photobiology of purified recombinant crystallins has not been studied. Here we examine photo-induced aggregation of purified recombinant mouse betaA3-crystallin (rbetaA3) and compare it with that of betaL-crystallins isolated from bovine lenses. METHODS: rbetaA3-Crystallin was expressed in baculovirus-infected Sf9 cells and purified by ion-exchange and gel-filtration chromatography. Protein solutions (pH 7.4) were irradiated at room temperature using a 308 nm excimer laser and light scattering was registered by attenuation of an unabsorbed beam of red light (670 nm). RESULTS: Irradiation of bovine alpha-crystallin, betaL-crystallin, rbetaA3-crystallin and gammaB-crystallin resulted in formation of insoluble aggregates with subsequent light scattering. Different slopes and threshold energies were observed for light scattering by each of these species. Sensitivity to ultraviolet irradiation induced light scattering as determined from threshold energies varied, with gamma-crystallins showing the greatest sensitivity, the betaL- and rbetaA3-crystallins showing an intermediate sensitivity and alpha-crystallins much less sensitive. Low doses (100 J/cm2) resulted in irreversible formation of water soluble oligomers but no insoluble aggregates as indicated by changes in light transmission. The photo-behavior of rbA3 was similar to mixed betaL-crystallin and different from that of alpha- and gamma-crystallins. CONCLUSIONS: Ultraviolet induced sensitivity of purified recombinant crystallins reflects that of mixed crystallin populations and should provide an indication of the pathogenicity of specific crystallin sequence changes associated with lens aging and hereditary cataract.

Animals↗
PubMed2002

The Scoring Clinical Index for Onychomycosis (SCIO index).

Onychomycosis is a common disease, and there are a number of factors that may affect the duration and dosage of treatment including the type of onychomycosis, the area and thickness of nail involvement, the age of the patient, and the location of the digit that is affected. We report a composite index, the Scoring Clinical Index for Onychomycosis (SCIO) that combines these factors to give an index of the overall severity of onychomycosis. The use of the SCIO may have treatment implications; by matching patients with similar SCIO scores, it may be possible to better compare the clinical response to therapy.

Adult↗
PubMed2000

Monomer-dimer equilibrium of normal and modified beta A3-crystallins: experimental determination and molecular modeling.

Beta- and gamma-crystallins are major protein constituents of the mammalian lens, where their stability and association into higher order complexes are critical for clarity and refraction. Two regions of the betagamma-crystallins have been suggested to modulate protein association, namely, the flexible N-terminal extensions and the intramolecular domain interfaces. The oligomeric state of wild-type recombinant murine betaA3-crystallin (rbetaA3) was compared to that of modified betaA3-crystallins with either an N-terminal deletion of residues 1 to 29 (rbetaA3tr) or with residues 114 to 123 of the interdomain linker replaced with the analogous linker from murine gammaB-crystallin (rbetaA3cp). All three proteins exhibited reversible monomer-dimer formation. The modifications to the N-terminus and domain linker resulted in tighter dimer formation as compared to wild-type protein as indicated by disassociation constants determined by sedimentation equilibrium: 6.62 x 10(-6) M (rbetaA3), 0.86 x 10(-6) M (rbetaA3cp), and 1.83 x 10(-7) M (rbetaA3tr). Homology modeling of betaA3-crystallins and solvation energy calculations also predicted tighter binding of the modified crystallins consistent with the centrifugation results. The findings suggest that under physiological conditions betaA3 crystallin exists in a dynamic equilibrium between monomeric and dimeric protein and that modification, especially to the N-terminal extension, can promote self-association.

Amino Acid Sequence↗
PubMed1998

Local microdomain structure in the terminal extensions of betaA3- and betaB2-crystallins.

PURPOSE: Although the crystal structures of the core domains of bovine betaB2-crystallin have been determined and those of other betagamma-crystallins modeled, the positions of the N- and C-termini are not resolvable by X-ray crystallography. Here we model the possible structural organization of the terminal arms of mouse betaA3- and betaB2-crystallins and test this model against the results of partial proteolysis. METHODS: The secondary structure of the terminal extensions was predicted by 3 different methods, one a nearest-neighbor method modified to use overlapping sequence tripeptides. Recombinant betaA3- and betaB2-crystallins were expressed using baculovirus vectors in S. frugiperda Sf9 cells. Crystallins were sequenced by the Edman degradation method. RESULTS: The N-terminal extension of betaB2-crystallin includes a series of hydrophilic residues from Q-11 to Q-9 which have high propensity of a helical conformation. The N-terminal arm of betaA3-crystallin is also predicted to have two helical segments, from Q-24 to E-20 and M-13 to A-12. Partial characterization of the baculovirus extract showed a thiol protease inhibited by leupeptin and E-64. As predicted by the model, recombinant betaB2-crystallin subjected to partial proteolysis was cleaved adjacent to the helical domain, while the N-terminal cleavage site in recombinant betaA3-crystallin was within 1 residue of an interhelical junction. Our model also predicts the products of partial proteolytic degradation of betaB2- and betaA3-crystallins from human, rat, bovine and chicken lenses incubated with the protease m-calpain. CONCLUSIONS: These results suggest the existence of local microdomain structures in the N- and C-terminal extensions of betaA3- and betaB2-crystallins, which appear to be more susceptible to proteolytic degradation in regions adjacent to these putative domains.

Amino Acid Sequence↗
PubMed1997

Association properties of betaB2- and betaA3-crystallin: ability to form dimers.

The beta-crystallins are a major constituent of the mammalian lens, where they associate into dimers, tetramers and higher order aggregates. Appropriate association of lens crystallins is important for lens transparency. To examine the associative properties of betaB2-crystallin, we have expressed mouse betaB2-crystallin using a baculovirus system. Recombinant mouse betaB2-crystallin has an estimated monomer molecular weight of 24 kDa by SDS-PAGE, appropriate immunoreactivity and appropriate secondary structure as assessed by circular dichroism analysis. The recombinant betaB2-crystallin associates into a homodimer with a weight average molecular mass of 39 kDa. The betaB2-crystallin homodimer has an estimated Kd of 5 x 10(-6) M, slightly greater than that of betaA3-crystallin, 0.8 x 10(-6) M. When recombinant betaB2-crystallin is combined with recombinant betaA3-crystallin, a heterodimer is formed within 10 min of incubation at room temperature. When equilibrium is reached in 4-6 h, approximately half of each crystallin associates into heterodimers. Subunit exchange between betaB2-crystallin and betaA3-crystallin occurs readily in the absence of any denaturing agents. Thus, rbetaA3-rbetaB2 heterodimer formation can occur under conditions similar to those found in the eye lens.

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↗