Biomedical subjects

O Ogun

Publications and source records attributed to O Ogun.

11 recordsLinked to original sources

PubMed2001

Enhanced crystallization of the Cys18 to Ser mutant of bovine gammaB crystallin.

The cysteine residues of the gamma crystallins, a family of ocular lens proteins, are involved in the aggregation and phase separation of these proteins. Both these phenomena are implicated in cataract formation. We have used bovine gammaB crystallin as a model system to study the role of the individual cysteine residues in the aggregation and phase separation of the gamma crystallins. Here, we compare the thermodynamic and kinetic behavior of the recombinant wild-type protein (WT) and the Cys18 to Ser (C18S) mutant. We find that the solubilities of the two proteins are similar. The kinetics of crystallization, however, are different. The WT crystallizes slowly enough for the metastable liquid-liquid coexistence to be easily observed. C18S, on the other hand, crystallizes rapidly; the metastable coexisting liquid phases of the pure mutant do not form. Nevertheless, the coexistence curve of C18S can be determined provided that crystallization is kinetically suppressed. In this way we found that the coexistence curve coincides with that of the WT. Despite the difference in the kinetics of crystallization, the two proteins were found to have the same crystal forms and almost identical X-ray structures. Our results demonstrate that even conservative point mutations can bring about dramatic changes in the kinetics of crystallization. The implications of our findings for cataract formation and protein crystallization are discussed.

Amino Acid Substitution↗

PubMed2001

Crystal cataracts: human genetic cataract caused by protein crystallization.

Several human genetic cataracts have been linked recently to point mutations in the gammaD crystallin gene. Here we provide a molecular basis for lens opacity in two genetic cataracts and suggest that the opacity occurs because of the spontaneous crystallization of the mutant proteins. Such crystallization of endogenous proteins leading to pathology is an unusual event. Measurements of the solubility curves of crystals of the Arg-58 to His and Arg-36 to Ser mutants of gammaD crystallin show that the mutations dramatically lower the solubility of the protein. Furthermore, the crystal nucleation rate of the mutants is enhanced considerably relative to that of the wild-type protein. It should be noted that, although there is a marked difference in phase behavior, there is no significant difference in protein conformation among the three proteins.

Cataract↗

PubMed2000

Molecular basis of a progressive juvenile-onset hereditary cataract.

In a recent paper, patients with a progressive juvenile-onset hereditary cataract have been reported to have a point mutation in the human gammaD crystallin gene (Stephan, D. A., Gillanders, E., Vanderveen, D., Freas-Lutz, D., Wistow, G., Baxevanis, A. D., Robbins, C. M., VanAuken, A., Quesenberry, M. I., Bailey-Wilson, J., et al. (1999) Proc. Natl. Acad. Sci. USA 96, 1008-1012). This mutation results in the substitution of Arg-14 in the native protein by a Cys residue. It is not understood how this mutation leads to cataract. We have expressed recombinant wild-type human gammaD crystallin (HGD) and its Arg-14 to Cys mutant (R14C) in Escherichia coli and show that R14C forms disulfide-linked oligomers, which markedly raise the phase separation temperature of the protein solution. Eventually, R14C precipitates. In contrast, HGD slowly forms only disulfide-linked dimers and no oligomers. These data strongly suggest that the observed cataract is triggered by the thiol-mediated aggregation of R14C. The aggregation profiles of HGD and R14C are consistent with our homology modeling studies that reveal that R14C contains two exposed cysteine residues, whereas HGD has only one. Our CD, fluorescence, and differential scanning calorimetric studies show that HGD and R14C have nearly identical secondary and tertiary structures and stabilities. Thus, contrary to current views, unfolding or destabilization of the protein is not necessary for cataractogenesis.

Adolescent↗

PubMed1998

Oligomerization and phase separation in globular protein solutions.

We have chemically crosslinked a globular protein, gamma IIIb-crystallin, to produce a system of well-defined oligomers: monomers, dimers, trimers and a mixture of higher n-mers. Gel electrophoresis, size exclusion chromatography, quasielastic light scattering spectroscopy, and electrospray ionization mass spectrometry were used to characterize the oligomers formed. The liquid-liquid phase separation boundaries of the various oligomers were measured. We find that at a given concentration the phase separation temperature strongly increases with the molecular weight of the oligomers. This phase behavior is very similar to previous findings for gamma II-crystallin, for which oxidation-induced oligomerization is accompanied by an increase in the phase separation temperature. These findings imply that for phase separation, the detailed changes of the surface properties of the proteins are less important than the purely steric effects of oligomerization.

Animals↗

PubMed1998

Aggregation in aqueous solutions of bovine lens gamma-crystallins: special role of gamma(s).

PURPOSE: To study the aggregation of bovine gamma-crystallins in aqueous solutions and the effect of gamma(s)-crystallin on the aggregation of other gamma-crystallins. METHODS: Aggregation in aqueous solutions of gamma(s)-, gammaII-, and gammaIVa-crystallin was monitored by quasielastic light scattering. Aggregation in mixtures of gamma(s)- and gammaII-crystallin, and gamma(s)- and gammaIVa-crystallin, was also monitored by light scattering. RESULTS: All of the gamma-crystallins studied formed large aggregates (or "megamers") in aqueous solutions. However, each protein differed in the relative rates of formation of megamers. Gamma(s)-crystallin formed megamers much more slowly than gammaII- and gammaIVa-crystallin. In solutions containing mixtures of gammaII and gamma(s), and gammaIVa and gamma(s), gamma(s)-crystallin significantly suppressed the aggregation of gammaII- and gammaIVa-crystallin. Megamerization seemed to be associated with thiol oxidation in these proteins. CONCLUSIONS: Gamma-crystallins undergo aggregation in which a small fraction of the proteins form a few large aggregates, whereas the larger proportion of the proteins remain monomeric. This suggests that the megamerization is preceded by an initial modification of the protein. The modification is associated with the thiol groups, and only such modified protein species participate in megamerization. The presence of gamma(s) significantly slows the megamerization of gammaII- and gammaIVa-crystallin. This fact, together with the previous finding that gamma(s) strongly reduces the phase separation temperatures of the gamma-crystallins, suggests that gamma(s)-crystallin plays an important role in suppressing the formation of light-scattering elements and helps maintain lens transparency.

Animals↗

PubMed1996

Phase separation in aqueous solutions of lens gamma-crystallins: special role of gamma s.

We have studied liquid-liquid phase separation in aqueous ternary solutions of calf lens gamma-crystallin proteins. Specifically, we have examined two ternary systems containing gamma s--namely, gamma IVa with gamma s in water and gamma II with gamma s in water. For each system, the phase-separation temperatures (Tph (phi)) alpha as a function of the overall protein volume fraction phi at various fixed compositions alpha (the "cloud-point curves") were measured. For the gamma IVa, gamma s, and water ternary solution, a binodal curve composed of pairs of coexisting points, (phi I, alpha 1) and (phi II, alpha II), at a fixed temperature (20 degrees C) was also determined. We observe that on the cloud-point curve the critical point is at a higher volume fraction than the maximum phase-separation temperature point. We also find that typically the difference in composition between the coexisting phases is at least as significant as the difference in volume fraction. We show that the asymmetric shape of the cloud-point curve is a consequence of this significant composition difference. Our observation that the phase-separation temperature of the mixtures in the high volume fraction region is strongly suppressed suggests that gamma s-crystallin may play an important role in maintaining the transparency of the lens.

Animals↗

PubMed1996

Pantethine inhibits the formation of high-Tc protein aggregates in gamma B crystallin solutions.

PURPOSE: Solutions of the bovine lens protein gamma B (or gamma II) crystallin at neutral pH in the absence of reducing agents, undergo a slow, partial conversion to a new protein species, gamma IIH. This species is an aggregate composed of an intermolecular, disulfide-crosslinked dimer (approximately equal to 32% of total protein by weight) and loosely associated dimers (approximately equal to 66%). gamma IIH has a phase separation temperature (Tph), at least 40 degrees C higher than that of native gamma II crystallin at any given protein concentration. In this paper we demonstrate that pantethine, a derivative of coenzyme A, inhibits the formation of gamma IIH. METHODS: gamma II crystallin solutions were incubated at pH 7.1 and room temperature with increasing amounts of pantethine. The Tph of the solutions was monitored as a function of incubation time. Corresponding to each Tph measurement, aliquots of each solution were analyzed by cation-exchange HPLC to determine the amount of gamma IIH formed. RESULTS: Incubation of gamma II crystallin with increasing amounts of pantethine lowers Tph and suppresses the formation of gamma IIH. With pantethine to protein mole ratios of 0.66, 1 and 2, the Tph of gamma II crystallin is lowered from 8 degrees C in the native protein, to 2 degrees C, -3 degrees C respectively, at a protein concentration of approximately equal to 200 mg/ml. The amount of gamma IIH accumulated decreases from approximately 25% in the native protein to 10%, 1% and 0% respectively in these pantethine-treated protein solutions. For complete suppression of the rise in Tph and inhibition of gamma IIH formation, a 2:1 mole ratio of pantethine to protein is required. CONCLUSIONS: We suggest that pantethine reacts with two cysteine residues of gamma IIH crystallin by forming a mixed disulfide, and effectively suppress protein aggregation and lowers Tph. This is due to the strong polar character of pantethine which reduces the net attractive interactions between the protein molecules.

Animals↗

PubMed1995

Oxidation of gamma II-crystallin solutions yields dimers with a high phase separation temperature.

Aqueous solutions of the bovine eye lens protein gamma II (or gamma B)-crystallin at neutral pH show a gradual increase in phase separation temperature, Tph, when allowed to stand for several weeks at room temperature without reducing agents. In a typical experiment, the Tph of the protein solution (218 mg/ml) increases from 2.5 +/- 1 degree C to 32.5 +/- 1 degree C after 21 days, and a new protein species, gamma IIH, is formed. The Tph of pure gamma IIH is at least 40 degrees C higher than that of pure gamma II. The average apparent hydrodynamic radius is 36 A for gamma IIH compared to 26 A for gamma II. The molecular mass of gamma IIH is approximately 41.5 kDa compared to 20 kDa for native gamma II. Therefore, gamma IIH is probably a dimer of gamma II crystallin. gamma IIH has a lower thiol content than gamma II and is not formed in the presence of dithiothreitol. We conclude that gamma IIH is a thiol oxidation product of gamma II-crystallin and is a dimer containing an intermolecular disulfide crosslink. Thus, some oxidative modifications of protein thiol groups lead to an increase in net attractive interactions between proteins. As a result, Tph increases and protein aggregates are formed. These two microscopic changes produce the increased light scattering associated with lens opacification.

Animals↗

PubMed1993

Suppression of phase separation in bovine gamma IV crystallin solutions: effect of modification by charged versus uncharged polar groups.

gamma IVa Crystallin, the ocular lens protein with the highest critical temperature (Tc) for phase separation, has been chemically modified with N-ethylmaleimide (NEM) at neutral pH. NEM, a polar but uncharged modifier reacts with the cysteine residues of the protein. A maximum of 80-85% of the thiol groups are modified and the phase separation temperature is lowered by about 10 degrees C at a protein concentration of 30 mg ml-1. The coexistence curve of the NEM-modified protein with nearly four out of the six cysteines modified was determined and compared with that obtained by modifying the protein with a charged reagent, N-bromoacetylethanolamine phosphate (NBAEP). NBAEP modifies both cysteine and methionine residues of gamma IVa crystallin. The results of these two modification studies indicate that for the same total degree of modification of the sulfur-containing residues, the suppression in Tc due to the charged NBAEP is at least twice as large as that due to the polar but uncharged NEM. In order to obtain some measure of the relative hydrophilicities of the two modifiers, we have estimated the free energies of hydration of the nonionic segments of NEM and NBAEP, using structural additivity schemes. We find that the intrinsic hydrophilicities of the nonionic segments of these two modifiers are nearly equal. Hence the much larger suppression of Tc resulting from NBAEP modification is most probably due to the strongly polar, doubly charged phosphate group.

Amino Acids↗

PubMed1992

Solid-liquid phase boundaries of lens protein solutions.

We report measurement of the solid-liquid phase boundary, or liquidus line, for aqueous solutions of three pure calf gamma-crystallin proteins: gamma II, gamma IIIa, and gamma IIIb. We also studied the liquidus line for solutions of native gamma IV-crystallin calf lens protein, which consists of 85% gamma IVa/15% gamma IVb. In all four proteins the liquidus phase boundaries lie higher in temperature than the previously determined liquid-liquid coexistence curves. Thus, over the range of concentration and temperature for which liquid-liquid phase separation occurs, the coexistence of a protein crystal phase with a protein liquid solution phase is thermodynamically stable relative to the metastable separated liquid phases. The location of the liquidus lines clearly divides these four crystallin proteins into two groups: those in which liquidus lines flatten at temperatures greater than 70 degrees C: gamma IIIa and gamma IV, and those in which liquidus lines flatten at temperatures less than 50 degrees C: gamma II and gamma IIIb. We have analyzed the form of the liquidus lines by using specific choices for the structures of the Gibbs free energy in solution and solid phases. By applying the thermodynamic conditions for equilibrium between the two phases to the resulting chemical potentials, we can estimate the temperature-dependent free energy change upon binding of protein and water into the solid phase.

Animals↗

PubMed1991

Suppression of phase separation in solutions of bovine gamma IV-crystallin by polar modification of the sulfur-containing amino acids.

The calf lens protein gamma IV-crystallin, a strong determinant of the net phase-separation temperature of the lens, was chemically modified with N-bromoacetylethanolamine phosphate. The phase-separation temperatures of solutions of the modified protein were measured and found to be dramatically reduced with respect to those of the native protein. At neutral pH the reagent alkylates only the cysteine and methionine residues and introduces a doubly charged phosphate anion at a maximum distance of 10-12 A from the sulfur atoms. At a protein concentration of 38 g/liter, and with 30% of the cysteines and 40% of the methionines alkylated, the phase-separation temperature is lowered from approximately 25 +/- 2 degrees C to approximately 12 +/- 2 degrees C. The ascending limbs of the coexistence curves for the native and modified proteins were determined at two different degrees of modification. The coexistence curve of the protein with 35% of the cysteines and 40% of the methionines modified shows that as protein concentration approaches the critical concentration of 289 g/liter, there is a much larger suppression of the critical temperature, from approximately 38 +/- 2 degrees C in the native protein to approximately 16 +/- 2 degrees C. Incubation of intact calf lenses in vitro with the reagent results in the suppression of the phase-separation temperature by 3-9 degrees C. These results are consistent with the view that the observed suppression in the critical temperature is due to an increase in the hydrophilicity of the protein in the vicinity of the sulfur-containing residues.

Animals↗