partitioning to determine the nature and the magnitude of the yS-PEG interactions and to quantitatively assess the effectiveness of PEG as a crystallizing agent for yS. 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 yS-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 yS molecules in the binary yS-water system are attractive. We compare the magnitude of the attraction found for yS with the results obtained for the other y-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 proteinwater systems than known previously.

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.

We have experimentally determined the coexistence surface characterizing the phase behavior of gammaD-betaB1-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 gammaD and betaB1 i.e., large difference of composition in the two coexisting phases. This demixing suggests that the energy of the gammaD-betaB1 attractive interaction is significantly smaller than the energy of the gammaD-gammaD attractive interaction. We also observed the lowering of the phase separation temperature upon increasing of the fraction of betaB1 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.

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.

The P23T mutation in the human gammaD-crystallin gene has in recent years been associated with a number of well known cataract phenotypes. To understand the molecular mechanism of lens opacity caused by this mutation, we expressed human gammaD-crystallin (HGD), the P23T mutant, and other related mutant proteins in Escherichia coli and compared the structures and thermodynamic properties of these proteins in vitro. The results show that the cataract-causing mutation P23T does not exhibit any significant structural change relative to the native protein. However, in marked contrast to the native protein, the mutant shows a dramatically lowered solubility. The reduced solubility results from the association of the P23T mutant to form a new condensed phase that contains clusters of the mutant protein. The monomer-cluster equilibrium is represented by a solubility curve in the phase diagram. When the solubility limit is exceeded, the mutant protein forms the condensed phase after a nucleation time of 10-20 min. We found that the solubility of the P23T mutant exhibits an inverse dependence on temperature, i.e., the protein clusters are increasingly soluble as the temperature of the solution decreases. The solubility of P23T can be substantially altered by the introduction of specific mutations at or in the immediate vicinity of residue 23. We examined the mutants P23S, P23V, P23TInsP24, and P23TN24K and found that the latter two mutations can restore the solubility of the P23T mutant. These findings may help develop a strategy for the rational design of small molecule inhibitors of this type of condensed phase.

Human betaB1-crystallin is a major eye-lens protein that undergoes in vivo truncation at the N-terminus with aging. By studying native betaB1 and truncated betaB1DeltaN41, which mimics an age-related in vivo truncation, we have determined quantitatively the effect of truncation on the oligomerization and phase transition properties of betaB1 aqueous solutions. The oligomerization studies show that the energy of attraction between the betaB1DeltaN41 proteins is about 10% greater than that of the betaB1 proteins. We have found that betaB1DeltaN41 aqueous solutions undergo two distinct types of phase transitions. The first phase transition involves an initial formation of thin rodlike assemblies, which then evolve to form crystals. The induction time for the formation of rodlike assemblies is sensitive to oligomerization. The second phase transition can be described as liquid-liquid phase separation (LLPS) accompanied by gelation within the protein-rich phase. We refer to this process as heterogeneous gelation. These two phase transitions are not observed in the case of betaB1 aqueous solutions. However, upon the addition of poly(ethylene glycol) (PEG), we observe heterogeneous gelation also for betaB1. Our PEG experiments allow us to estimate the difference in phase separation temperatures between betaB1 and betaB1DeltaN41. This difference is consistent with the increase in energy of attraction found in our oligomerization studies. Our work suggests that truncation is a cataractogenic modification since it favors protein condensation and the consequent formation of light scattering elements, and highlights the importance of the N-terminus of betaB1 in maintaining lens transparency.

RESULTS. All of the y-crystallins studied formed large aggregates (or "megamers") in aqueous solutions. However, each protein differed in the relative rates of formation of megamers. ysCrystallin formed megamers much more slowly than y,,and yIVa-crystallin. In solutions containing mixtures of y,, and ys, and y,Va and ys, ys-crystallin significantly suppressed the aggregation of y,,and yIV:i-crystallin. Megamerization seemed to be associated with thiol oxidation in these proteins.

Several human cataracts have been linked to mutations in the γ crystallin gene. One of these is the aculeiform cataract, which is caused by an R58H mutation in γD 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 γ crystallins.

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.

We have studied the effect of polyethylene glycol (PEG) on the liquid-liquid phase separation (LLPS) of aqueous solutions of bovine gammaD-crystallin (gammaD), 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 gammaD-PEG-water systems are used to successfully predict the location of the liquid-liquid phase boundary of the binary gammaD-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.

The cysteine residues of the γ 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 γB crystallin as a model system to study the role of the individual cysteine residues in the aggregation and phase separation of the γ 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.

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.