We have determined the coexistence curves (plots of phase-separation temperature T versus protein concentration C) for aqueous solutions of purified calf lens proteins. The proteins studied, calf y'Ila-, yIlb-, and yIVacrystallin, have very similar amino acid sequences and threedimensional structures. Both ascending and descending limbs of the coexistence curves were measured. We find that the coexistence curves for each of these proteins and for yIcrystallin can be fit, near the critical point, to the function W(Cc -C)/CJI = A[(Tc -T)/TCJ, where fi = 0.325, Cc is the critical protein concentration in mg/ml, T, is the critical temperature for phase separation in K, and A is a parameter that characterizes the width of the coexistence curve. We find that A andCc are approximately the same for all four coexistence curves (A = 2.6 +-0.1, Cc = 289 ± 20 mg/ml), but that Tc is not the same. For yIH-and yIIIb-crystallin, Tc7-5°C, whereas for yIIa-and yIVa-crystallin, Tc 38C. By comparing the published protein sequences for calf, rat, and human y-crystallins, we postulate that a few key amino acid residues account for the division of y-crystallins into low-Tc and high-Tc groups.The y-crystallins constitute a family of highly homologous mammalian lens proteins (1-4). Concentrated aqueous solutions of y-crystallins (5-8) exhibit the phenomena of binaryliquid-phase separation (9-11), also known as coacervation (12). These solutions separate into two coexisting liquid phases of unequal protein concentration at temperatures less than the critical temperature for phase separation Tc. From previous studies (5,7,8), it is known that location of the coexistence curve depends sensitively on the amino acid sequence of the crystallin molecule. Two distinct groups of y-crystallins have been identified in rat (7) and human (8) lenses: high-Tc crystallins and low-Tc crystallins. In these rat and human studies, the precise values of Tc for each crystallin, though inferred from the data, were not determined explicitly. For the high-Tc crystallins, only the ascending limb of the coexistence curves was measured. For the low-Tc crystallins, only an upper bound for the Tc values was established.In this paper, we report on measurements of coexistence curves for three purified calf y-crystallins-yIIIa, yIIIb, and yIVa-[in Table 2 we indicate the current nomenclature for mammalian y-crystallins (2)] and for the native calf y-crystallin mixture yIV. We have determined both the ascending and descending limbs of each coexistence curve. This information enables us to characterize the coexistence curves in detail and to determine explicitly the values of the critical concentration Cc and the critical temperature Tc for each protein. Such detailed analysis of y-crystallin phase separation, which requires gram quantities of purified protein, has been performed only for calf yII (6).We find that the purified calf y-crystallins, in accord with the purified rat and human y-crystallins, fall into two distinct groups: high-Tc (Tc > 350C) proteins, ...

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Several human genetic cataracts have been linked recently to point mutations in the ␥D 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 ␥D 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.H uman ␥D crystallin is a member of a highly homologous family of mammalian lens proteins called the ␥ crystallins (1). Together with the ␣ and ␤ crystallins, these proteins are essential for maintaining lens transparency. However, the ␥ crystallins differ from the ␣ and ␤ crystallins in one important respect: the interactions between the ␥ crystallins are attractive (2). This feature reduces the osmotic pressure in the lens, but it also makes the ␥ crystallins more susceptible to aggregation and phase separation, phenomena that diminish the homogeneity of the lens and lead to cataract (3). Yet, despite these attractive interactions, the ␥ crystallins remain soluble for many years at high concentrations and with little turnover, maintaining the proper refractive index gradient of the lens (4).It is well known that random mutations in proteins dramatically affect their solubility (5). In this article, we show that the Arg-58 to His (R58H) mutant [linked to the aculeiform cataract (Fig. 1a; ref. 6)] and the Arg-36 to Ser (R36S) mutant [linked to another form of genetic (congenital) cataract ( Fig. 1b; ref. 7)] are much less soluble than the wild-type human ␥D crystallin protein (HGD). We also show that these mutants are more prone to crystallize than the wild-type. Indeed, Kmoch et al. (7) recently extracted crystals of the R36S mutant from the eye of a young patient. To determine the mechanism of cataract formation caused by these mutations, we compared the conformation, stability, and phase behavior of the recombinant HGD, R58H, and R36S proteins in solution. Materials and MethodsCloning, Expression, and Isolation of Proteins. Recombinant human ␥D crystallin was prepared by the amplification of the coding sequence from a human fetal lens cDNA library as detailed (8). Overexpression of ␥D crystallin, and isolation and purification of the protein, were all done according to procedures as described (8). Mutant proteins were prepared as follows.(i) The R58H mutant. To introduce a histidine mutation in place of Arg-58, the following oligonucleotide primers were made: 5Ј-CCAGTACTTCCTGCACCGCGGCGACTATGC-3Ј as the forward primer and 5Ј-GCATAGTCGCCGCGGTGCAG-GAAGTACTGG-3Ј as the reverse prime...

We have expressed recombinant wild-type human ␥D 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 thiolmediated 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.I n the hereditary, juvenile-onset cataract described by Stefan et al. (1), the lens, which is clear at birth, develops punctate opacities progressively, such that by two years of age the cataract is readily detectable, and matures by early childhood or adolescence. The punctate opacities seen in this cataract are in the nucleus and inner cortex, regions of the lens that are enriched in the ␥-crystallins. In the human lens, only two members of the ␥-crystallin family, ␥C and ␥D, are expressed in appreciable amounts, and only ␥D crystallin continues to be expressed until late childhood (2, 3). In affected individuals, a single point mutation has been identified in the ␥D crystallin gene that corresponds to the substitution of Arg-14 by a Cys. The identification of this mutation and the parallel between the time course of the pathology and the physiological expression of human ␥D crystallin strongly implicate the Arg-14 3 Cys mutant of ␥D in the development of this cataract. However, the molecular mechanism invoked to explain the observed opacity has been speculative (1).In the past, it has not been possible to conduct detailed studies on human ␥D crystallin because of the difficulty of obtaining sufficient quantities of pure protein from young, normal human lenses (4). Therefore, to characterize the normal protein thoroughly and investigate the mechanism by which the Arg-14 3 Cys mutation in ␥D could lead to cataract, we cloned and expressed human ␥D crystallin and its Arg-14 3 Cys mutant in Escherichia coli. Both the wild-type recombinant ␥D crystallin (HGD) and its Arg-14 3 Cys mutant (R14C) folded efficiently in E. coli and accumulated as soluble proteins. We isolated and purified the HGD and R14C proteins and determined their solution properties. Our results suggest that the disulfidecrosslinked oligomerization of R14C is responsible for the observed cataract. Furthermore, such oligomerization occurs without significant change in protein structure, conformation, and stability. Materials and MethodsCloning, Expression, and Isolation of Proteins. The human ␥D crystallin coding sequence was amplif...

We have studied the effect of polyethylene glycol (PEG) on the liquid-liquid phase separation (LLPS) of aqueous solutions of bovine ␥D-crystallin (␥D), 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 ␥D-PEGwater systems are used to successfully predict the location of the liquid-liquid phase boundary of the binary ␥D-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.PEG ͉ ternary mixtures ͉ solubility ͉ partitioning P olyethylene glycol (PEG) is a hydrophilic nonionic polymer used in many biochemical and industrial applications. Due to its nontoxic character, this chemical can be found in cosmetics, food, and pharmaceutical products. The mild action of PEG on the biological activity of cell components explains the success of this polymer in biotechnological applications. PEG is commonly used for liquid-liquid partitioning and precipitation of biomacromolecules (1, 2). In protein crystallography, PEG is considered the most successful precipitating agent for the production of protein crystals, the crucial step for the determination of the molecular structure of a protein. All these applications make PEG by far the most widely used polymer in aqueous solutions of biological molecules (1, 3).Due to the extensive practical use of PEG as a precipitating agent for proteins, it is of fundamental importance to understand protein-protein and protein-PEG interactions in protein-PEGwater ternary systems. These interactions are often described in terms of a depletion force, which arises because the polymer is depleted in the region between adjacent proteins (4, 5). Depletion force models have been successful in describing the effect of nonadsorbing polymers on colloidal suspensions (6-10).Protein-PEG-water solutions have been investigated by several techniques (4,5,(11)(12)(13)(14). Small-angle x-ray-(4) and lightscattering (5) measurements generally confirmed that total protein-protein interactions can be described in terms of depletion effects. The microscopic interpretation used in the above studies is derived from colloid-polymer models. To model the effect of PEG, a depletion component is added to the original protein-protein pair interaction potential. This microscopic modeling, however, does not provide information about the actual protein-PEG intera...

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.

The self-assembly of helical ribbons is examined in a variety of multicomponent enantiomerically pure systems that contain a bile salt or a nonionic detergent, a phosphatidylcholine or a fatty acid, and a steroid analog of cholesterol. In almost all systems, two different pitch types of helical ribbons are observed: high pitch, with a pitch angle of 54 ؎ 2°, and low pitch, with a pitch angle of 11 ؎ 2°. Although the majority of these helices are right-handed, a small proportion of left-handed helices is observed. Additionally, a third type of helical ribbon, with a pitch angle in the range 30-47°, is occasionally found. These experimental findings suggest that the helical ribbons are crystalline rather than liquid crystal in nature and also suggest that molecular chirality may not be the determining factor in helix formation. The large yields of helices produced will permit a systematic investigation of their individual kinetic evolution and their elastic moduli.Interest in molecular self-assembly of helical structures is driven by both technological and medical applications. Helices are often precursors in the growth of tubules (1-4), which can be used as a controlled release system for drug delivery in medicine and as templates for microelectronics and magnetic applications (5). The morphology of the tubules must be rationally optimized for each application. Therefore it is important to understand the role of the various constituent molecules in the formation of these structures.Helical ribbons have been observed in a variety of systems composed of chiral amphiphiles. Although the diameters and lengths of the helical structures varied from system to system, the pitch angle observed in all systems was either 45°(6-8) or Ϸ60°(9-12). In almost all cases, helical ribbons in enantiomerically pure systems were either right-or left-handed (6,8,13). Recently, however, Thomas et al. studied an enantiomerically pure phosphonate analogue of 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine and related compounds, which self-assembled into a mixture of right-and left-handed helical ribbons (10, 11).A biologically important system in which helical ribbons form is model bile, consisting of a mixture of three types of chiral molecules in water: a bile salt, a phosphatidylcholine, and cholesterol (4, 14-21). Helical ribbons are metastable intermediates in the process of cholesterol crystallization in bile (2,19,20), which precedes cholesterol gallstone formation (4,(18)(19)(20)(21)(22)(23). In contrast to all other systems studied, two pitch types of helical ribbons are observed in bile: high pitch, with a pitch angle of 54 Ϯ 2°, and low pitch, with a pitch angle of 11 Ϯ 2°. To date, the production of these two helical pitch types has been thought to be a property unique to model biles. Indeed, previous work showed that all three components of model bile are required for helical ribbons to form. In the absence of the phosphatidylcholine, only needle-like crystals form (4, 21), whereas without the bile salt only...

The P23T mutant of human ␥D-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.cataract ͉ lens ͉ protein phase diagram ͉ quasielastic light scattering H uman ␥D-crystallin (HGD) is an important member of the ␥-crystallin family of proteins found in the human lens. Mutations in HGD in particular have been associated with a number of childhood cataracts (1-4). Recently, the P23T mutant of HGD has been associated with coralliform, cerulean, and fasciculiform cataract phenotypes (refs. 1 and 2 and references therein). Mutation at site 23 of HGD is only one of a number of single point mutations, including the R14C, R58H, and R36S mutations, that occur on the CRGD gene and that have been linked with early-onset cataract disease (5-9). Physicochemical characterization of the mutant proteins shows why these changes result in the formation of either covalently linked aggregates in the case of R14C (5, 6) or crystals in the case of the R58H and R36S mutants (7-9). The P23T mutation results in decreased solubility of the protein, leading to protein aggregation and light scattering, and hence to lens opacity. However, the aggregates are not covalently linked, since the aggregation process is completely reversible with temperature.Formation of protein aggregates is a common motif in many ''condensation diseases,'' which include cataract (10), sickle-cell anemia (11,12), and Alzheimer's disease (13,14), as well as other amyloid diseases such as diabetes and Parkinson's disease (15,16). These examples highlight the importance of understanding the processes that lead to the formation of condensed protein phases under physiological conditions. In particular, protein condensation diseases resulting from a single amino acid substitution provide favorable conditions for biophysical analysis because such substitutions produce a change in the interaction potential between the proteins but may not necessarily result in significant structural changes to the protein itself. T...