Olutayo Ogun

Crystal cataracts: Human genetic cataract caused by protein crystallization

Several human genetic cataracts have been linked recently to point mutations in the γD 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 γD 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.

High-resolution X-ray Crystal Structures of Human γD Crystallin (1.25 Å) and the R58H Mutant (1.15 Å) Associated with Aculeiform Cataract

Several human cataracts have been linked to mutations in the gamma crystallin gene. One of these is the aculeiform cataract, which is caused by an R58H mutation in gammaD crystallin. We have shown previously that this cataract is caused by crystallization of the mutant protein, which is an order of magnitude less soluble than the wild-type. Here, we report the very high-resolution crystal structures of the mutant and wild-type proteins. Both proteins crystallize in the same space group and lattice. Thus, a strict comparison of the protein-protein and protein-water intermolecular interactions in the two crystal lattices is possible. Overall, the differences between the mutant and wild-type structures are small. At position 58, the mutant protein loses the direct ion-pair intermolecular interaction present in the wild-type, due to the differences between histidine and arginine at the atomic level; the interaction in the mutant is mediated by water molecules. Away from the mutation site, the mutant and wild-type lattice structures differ in the identity of side-chains that occupy alternate conformations. Since the interactions in the crystal phase are very similar for the two proteins, we conclude that the reduction in the solubility of the mutant is mainly due to the effect of the R58H mutation in the solution phase. The results presented here are also important as they are the first high-resolution X-ray structures of human gamma crystallins.

Effect of polyethylene glycol on the liquid-liquid phase transition in aqueous protein solutions.

We have studied the effect of polyethylene glycol (PEG) on the liquid–liquid phase separation (LLPS) of aqueous solutions of bovine γD-crystallin (γD), a protein in the eye lens. We observe that the phase separation temperature increases with both PEG concentration and PEG molecular weight. PEG partitioning, which is the difference between the PEG concentration in the two coexisting phases, has been measured experimentally and observed to increase with PEG molecular weight. The measurements of both LLPS temperature and PEG partitioning in the ternary γD-PEG-water systems are used to successfully predict the location of the liquid–liquid phase boundary of the binary γD-water system. We show that our LLPS measurements can be also used to estimate the protein solubility as a function of the concentration of crystallizing agents. Moreover, the slope of the tie-lines and the dependence of LLPS temperature on polymer concentration provide a powerful and sensitive check of the validity of excluded volume models. Finally, we show that the increase of the LLPS temperature with PEG concentration is due to attractive protein–protein interactions.

Altered phase diagram due to a single point mutation in human gammaD-crystallin.

The P23T mutant of human γD-crystallin (HGD) is associated with cataract. We have previously investigated the solution properties of this mutant, as well as those of the closely related P23V and P23S mutants, and shown that although mutations at site 23 of HGD do not produce a significant structural change in the protein, they nevertheless profoundly alter the solubility of the protein. Remarkably, the solubility of the mutants decreases with increasing temperature, in sharp contrast to the behavior of the native protein. This inverted solubility corresponds to a strong increase in the binding energy with temperature. Here we have investigated the liquid–liquid coexistence curve and the diffusivity of the P23V mutant and find that these solution properties are unaffected by the mutation. This means that the chemical potentials in the solution phase are essentially unaltered. The apparent discrepancy between the interaction energies in the solution phase, as compared with the solid phase, is explicable in terms of highly anisotropic interprotein interactions, which are averaged out in the solution phase but are fully engaged in the solid phase.

Static structure factor and collective diffusion of globular proteins in concentrated aqueous solution

We report our measurement of the time average and the temporal autocorrelation function of the intensity of light scattered by the highly monomeric globular protein, bovine γII‐crystallin, in aqueous solution as a function of wave number q, protein volume fraction φ, and temperature T. The time average intensity data is used to obtain the q→0 limit of the static structure factor S(φ,T), as a function of φ and T. We show that S(φ,T) may be well characterized by modeling the proteins as interacting through the Baxter adhesive hard sphere pair interaction potential. The temporal autocorrelation function data is used to determine the collective diffusion coefficient D(φ,T) of the proteins as a function of φ and T. We then obtain the experimental hydrodynamic factor H(φ,T)≡S(φ,T)[D(φ,T)/D0(T)], where D0(T) is the diffusion coefficient of the individual proteins in the φ→0 limit. We find that H exhibits a different φ‐dependence at low (φ≤0.016) and high (φ≳0.02) protein volume fractions. In the low φ domain...

Observation of liquid–liquid phase separation for eye lens γS-crystallin

γS-crystallin (γS) is an important human and bovine eye lens protein involved in maintaining the transparency of the eye. By adding small amounts of polyethylene glycol (PEG) to the binary aqueous bovine γS solutions, we have observed liquid–liquid phase separation (LLPS) at −8°C and revealed that, in the binary γS–water system, this phase transition would occur at −28°C. We have measured both the effect of PEG concentration on the LLPS temperature and protein/PEG partitioning between the two liquid coexisting phases. We use our measurements of protein/PEG partitioning to determine the nature and the magnitude of the γS-PEG interactions and to quantitatively assess the effectiveness of PEG as a crystallizing agent for γS. We use our measurements of LLPS temperature as a function of protein and PEG concentration to successfully determine the location of the critical point for the binary γS-water system. This phase transition cannot be observed in the absence of PEG because it is inaccessible due to the freezing of the system. Our findings indicate that the effective interactions between γS molecules in the binary γS–water system are attractive. We compare the magnitude of the attraction found for γS with the results obtained for the other γ-crystallins for which the critical temperature is located above the freezing point of the system. This work suggests that PEG can be used to reveal the existence of LLPS for a much wider range of binary protein–water systems than known previously.

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.

Phase behavior of mixtures of human lens proteins Gamma D and Beta B1

We have experimentally determined the coexistence surface characterizing the phase behavior of γD-βB1-water ternary solutions. The coexistence surface fully describes the solution conditions, i.e., temperature, protein concentration, and protein composition, at which liquid-liquid phase separation occurs in a ternary solution. We have observed a significant demixing of γD and βB1 i.e., large difference of composition in the two coexisting phases. This demixing suggests that the energy of the γD-βB1 attractive interaction is significantly smaller than the energy of the γD-γD attractive interaction. We also observed the lowering of the phase separation temperature upon increasing of the fraction of βB1 in solution. We provide a theoretical analysis of our experimental data, which enables a quantitative description of our principal experimental findings. In this way, we have evaluated the magnitude and temperature dependence of the relevant interprotein interaction energies. Our findings provide insight into the factors essential for maintaining lens proteins in a single homogeneous phase, thereby enabling lens transparency.

Pathological crystallization of human immunoglobulins.

Condensation of Igs has been observed in pharmaceutical formulations and in vivo in cases of cryoglobulinemia. We report a study of monoclonal IgG cryoglobulins overexpressed by two patients with multiple myeloma. These cryoglobulins form crystals, and we measured their solubility lines. Depending on the supersaturation, we observed a variety of condensate morphologies consistent with those reported in clinical investigations. Remarkably, the crystallization can occur at quite low concentrations. This suggests that, even within the regular immune response to infections, cryoprecipitation of Ig can be possible.

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.