Structure and stability of γ-crystallins. I. Spectroscopic evaluation of secondary and tertiary structure in solution

Mandal, Krishnagopal; Bose, S. K.; Chakrabarti, Buddhapriya; Siezen, Roland J.

doi:10.1016/0167-4838(85)90327-9

Cited by 47 publications

(37 citation statements)

References 42 publications

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“…4. The max values for HGD and R14C are 325 nm and 329 nm, respectively, indicating that the tryptophan residues in both proteins are buried (27). These values are well within the range of those observed for all native bovine ␥-crystallins ( max from 324-335 nm; ref.…”

Section: Resultssupporting

confidence: 81%

“…In general, ellipticities in the near-UV region arise from the tertiary structure of proteins. For the ␥-crystallins, this CD region is dominated by the four invariant tryptophan residues (27). In Fig.…”

Section: Resultsmentioning

confidence: 92%

“…The fluorescence emission spectrum of ␥-crystallins arises mainly from the four buried tryptophan residues that are invariant in all members of this family (27). The tryptophan residues, two in each domain, are excellent reporter groups that monitor the structural integrity of the protein.…”

Section: Resultsmentioning

confidence: 99%

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Molecular basis of a progressive juvenile-onset hereditary cataract

Pande

Asherie

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

131

184

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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...

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Section: Resultssupporting

confidence: 81%

Section: Resultsmentioning

confidence: 92%

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Molecular basis of a progressive juvenile-onset hereditary cataract

Pande

Asherie

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

131

184

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“…The additional minimum at 206-208 nm observed for the isolated N-terminal domain corresponds to a shoulder detectable in the spectrum of intact yB. This shoulder has been ascribed to contributions of aromatic side chains (Mandal et al, 1985). Neither near-UV CD nor fluorescence give any indication of significant structural changes or misfolding of 1 2 3 4 5 6 7 8 9 Gill and von Hippel (1989).…”

Section: Resultsmentioning

confidence: 99%

Mutational analysis of hydrophobic domain interactions in γB‐crystallin from bovine eye lens

1997

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AbstractyB-crystallin is a monomeric member of the By-superfamily of vertebrate eye lens proteins. It consists of two similar domains with all+ Greek key topology associating about an approximate two-fold axis. At pH 2, with urea as the denaturant, the domains show independent equilibrium unfolding transitions, suggesting different intrinsic stabilities. Denaturation experiments using recombinant one-or two-domain proteins showed that the N-terminal domain on its own exhibits unaltered intrinsic stability but contributes significantly to the stability of its C-terminal partner.It has been suggested that docking of the domains is determined by a hydrophobic interface that includes phenylalanine at position 56 of the N-terminal domain. In order to test this hypothesis, F56 was substituted by site-directed mutagenesis in both complete yB-crystallin and its isolated N-terminal domain. All mutations destabilize the N-terminal domain to about the same extent but affect the C-terminal domain in a different way. Replacement by the small alanine side chain or the charged aspartic acid residue results in a significant destabilization of the C-terminal domain, whereas the more bulky tryptophan residue causes only a moderate decrease in stability. In the mutants F56A and F56D, equilibrium unfolding transitions obtained by circular dichroism and intrinsic fluorescence differ, suggesting a more complex denaturation behavior than the one observed for yB wild type. These results confirm how mutations in one crystallin domain can affect the stability of another when they occur at the interface. The results strongly suggest that size, hydrophobicity, and optimal packing of amino acids involved in these interactions are critical for the stability of yB-crystallin.

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“…S2A. γ-crystallins contain four buried tryptophan residues, two in each domain, which are excellent reporters of the overall structural integrity of the protein (27). Fig.…”

Section: Resultsmentioning

confidence: 99%

Cataract-associated mutant E107A of human γD-crystallin shows increased attraction to α-crystallin and enhanced light scattering

Banerjee¹,

Pande²,

Patrosz

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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Several point mutations in human γD-crystallin (HGD) are now known to be associated with cataract. So far, the in vitro studies of individual mutants of HGD alone have been sufficient in providing plausible molecular mechanisms for the associated cataract in vivo. Nearly all the mutant proteins in solution showed compromised solubility and enhanced light scattering due to altered homologous γ-γ crystallin interactions. In sharp contrast, here we present an intriguing case of a human nuclear cataract-associated mutant of HGD-namely Glu107 to Ala (E107A), which is nearly identical to the wild type in structure, stability, and solubility properties, with one exception: Its pI is higher by nearly one pH unit. This increase dramatically alters its interaction with α-crystallin. There is a striking difference in the liquid-liquid phase separation behavior of E107A-α-crystallin mixtures compared to HGD-α-crystallin mixtures, and the light-scattering intensities are significantly higher for the former. The data show that the two coexisting phases in the E107A-α mixtures differ much more in protein density than those that occur in HGD-α mixtures, as the proportion of α-crystallin approaches that in the lens nucleus. Thus in HGD-α mixtures, the demixing of phases occurs primarily by protein type while in E107A-α mixtures it is increasingly governed by protein density. Analysis of these results suggests that the cataract due to the E107A mutation could result from the instability caused by the altered attractive interactions between dissimilar proteins -i.e., heterologous γ-α crystallin interactions-primarily due to the change in surface electrostatic potential in the mutant protein.T he vertebrate lens contains three families of constituent proteins, the α-, β-, and γ-crystallins, closely packed such that the total protein concentration in the central region of the lens, i.e., nucleus, exceeds 400 mg∕mL (1). Normally, the close protein packing ensures that short-range order is maintained within the fiber cell, such that fluctuations in the refractive index that cause light scattering are minimized, and the lens is transparent (2, 3). Disruption of short-range order associated with aging, altered environment, or mutations in crystallins can cause increased light scattering and cataract.Cataract-associated mutations in the crystallin genes are known to occur in all three crystallin families (1, 4) and show a variety of phenotypes. Over the last decade, we have focused on the point mutations occurring in human γD-crystallin (HGD), a member of the γ-crystallin family expressed at high levels along with γC-and γS-crystallins in the human lens (5). By comparing the properties of several mutants of HGD in solution with those of the wild type, we have identified specific changes in the homologous (i.e., γ-γ interactions) that led to the formation of distinct condensed phases (6, 7) and accounted for the increased light scattering due to these mutations (8-13). These studies revealed a class of γ-crystallin mutations in which smal...

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Structure and stability of γ-crystallins. I. Spectroscopic evaluation of secondary and tertiary structure in solution

Cited by 47 publications

References 42 publications

Molecular basis of a progressive juvenile-onset hereditary cataract

Molecular basis of a progressive juvenile-onset hereditary cataract

Mutational analysis of hydrophobic domain interactions in γB‐crystallin from bovine eye lens

Cataract-associated mutant E107A of human γD-crystallin shows increased attraction to α-crystallin and enhanced light scattering

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