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...