Human gammaD-crystallin Research Articles

An ultraviolet-driven rescue pathway for oxidative stress to eye lens protein human gamma-D crystallin

Human gamma-D crystallin (HGD) is a major constituent of the eye lens. Aggregation of HGD contributes to cataract formation, the leading cause of blindness worldwide. It is unique in its longevity, maintaining its folded and soluble state for 50-60 years. One outstanding question is the structural basis of this longevity despite oxidative aging and environmental stressors including ultraviolet radiation (UV). Here we present crystallographic structures evidencing a UV-induced crystallin redox switch mechanism. The room-temperature serial synchrotron crystallographic (SSX) structure of freshly prepared crystallin mutant (R36S) shows no post-translational modifications. After aging for nine months in the absence of light, a thiol-adduct (dithiothreitol) modifying surface cysteines is observed by low-dose SSX. This is shown to be UV-labile in an acutely light-exposed structure. This suggests a mechanism by which a major source of crystallin damage, UV, may also act as a rescuing factor in a finely balanced redox system.

Catalysis of Aggregation by Interface Opening and Disulfide Exchange in Cataract-Associated Variants of Human Gamma-D Crystallin

Catalysis of Aggregation by Interface Opening and Disulfide Exchange in Cataract-Associated Variants of Human Gamma-D Crystallin

Disulfide Exchange and Self-Catalyzed Aggregation in Cataract-Associated Human Gamma-D Crystallin

Disulfide Exchange and Self-Catalyzed Aggregation in Cataract-Associated Human Gamma-D Crystallin

An Internal Disulfide Locks a Misfolded Aggregation-Prone Intermediate in Cataract-Linked Mutants of Human Gamma-D Crystallin

Considerable mechanistic insight has been gained into amyloid aggregation; however, a large class of non-amyloid protein aggregates are considered 'amorphous,' and in most cases little is known about their mechanisms. Amorphous aggregation of {\gamma}-crystallins in the eye lens causes a widespread disease of aging, cataract. We combined simulations and experiments to study the mechanism of aggregation of two {\gamma}D-crystallin mutants, W42R and W42Q - the former a congenital cataract mutation, and the latter a mimic of age-related oxidative damage. We found that formation of an internal disulfide was necessary and sufficient for aggregation under physiological conditions. Two-chain all-atom simulations predicted that one non-native disulfide in particular, between Cys32 and Cys41, was likely to stabilize an unfolding intermediate prone to intermolecular interactions. Mass spectrometry and mutagenesis experiments confirmed the presence of this bond in the aggregates and its necessity for oxidative aggregation under physiological conditions in vitro. Mining the simulation data linked formation of this disulfide to extrusion of the N-terminal \b{eta}-hairpin and rearrangement of the native \b{eta}-sheet topology. Specific binding between the extruded hairpin and a distal \b{eta}-sheet, in an intermolecular chain reaction similar to domain swapping, is the most probable mechanism of aggregate propagation.

An Investigation of the Atomic Structure of Cataract-Forming Mutant Gamma-D-Crystallin Aggregates Formed under Distinct Environmental Conditions

Human gamma-D-crystallin (HGD) is an extremely soluble (>400mg/mL in vivo) and stable protein that under certain conditions succumbs to protein aggregation, which is associated with cataract formation. Cataracts are the leading cause of blindness globally. Both environmental and genetic factors enable cataract formation by disrupting the solubility of HGD. Several chemical modifications are thought to be linked to loss of solubility and precipitation of HGD. The P23T mutant of HGD is genetically linked to three distinct congenital cataracts. It is crucial to develop a mechanistic understanding of how the reduced solubility of P23T HGD is caused by chemical and/or structural changes. We performed structural and chemical analyses on aggregated or aggregation-prone HGD to gain a molecular understanding of the aggregation mechanism, as well as the nature and location of chemical modifications.We find P23T HGD is aggregation prone, but forms qualitatively different aggregates as a function of the environmental conditions. Among other factors, we examined the impact of pH and UV irradiation on aggregation and evaluated the resulting aggregates by Electron Microscopy (EM), X-ray powder diffraction, Mass Spectrometry (MS), and multidimensional magic-angle-spinning (MAS) solid-state Nuclear Magnetic Resonance (NMR). Distinct conformations of HGD were observed in the aggregated state by X-ray powder diffraction and EM, and chemical changes were observed by MS. Through comparison to the NMR chemical shifts of the protein in the soluble and aggregated states, we identified structural changes in a residue and site-specific fashion. Our data reveal that aggregation conditions not only affect the macroscopic appearance of the aggregates, but also have a dramatic impact on the aggregates’ internal atomic structure. Therefore, distinct molecular mechanisms for crystallin aggregation exist.

Aggregation of Trp>Glu Mutants of the Human Gamma-D Crystallin: A Model for Hereditary or UV-Induced Cataract

Cataract is a protein misfolding disorder resulting from formation of light-scattering aggregates of proteins from the crystallin family. These long-lived proteins account for 90% of all protein in the human eye lens. The γD crystallin consists of two symmetrical domains, each of which has a duplicated Greek key fold. Maintaining this topologically complex native fold of the γD crystallin is essential for lens transparency. A number of point mutants in the γD crystallin gene cause early-onset cataract. UV-B exposure accelerates cataract onset, and UV irradiation of purified wild-type γD results in aggregation in vitro. A distinctive feature of the Greek key fold is the presence of highly conserved buried tryptophans. Replacements of tryptophan by charged glutamate groups may represent a model of UV-induced photodamage -- introduction of a charged group into the hydrophobic core generating “denaturation from within.” We show that such Trp>Glu mutants can display vastly increased aggregation propensity under physiological conditions in vitro. Furthermore, a striking property of these mutants is their ability to drive the wild-type protein into the aggregated state. Domain swapping mechanisms can account for this aggregation behavior.

Structure, Dynamics and Surface Hydrophobicity of the Cataract-Associated Mutant, Pro23Thr of Human Gamma D-crystallin: Molecular Basis of Cataract Formation

The cataract-associated Pro23Thr (P23T) mutation in human gammaD-crystallin (HGD) is geographically widespread - thus there is considerable interest in determining the molecular basis of opacity. In an earlier study (1), we found that the mutant showed markedly lowered, retrograde solubility compared to wild-type HGD, leading to the conclusion that the aggregation of P23T was mediated by hydrophobic protein-protein interactions. Subsequently, using NMR (2) and a binding assay with the fluorescent dye Bis-ANS (3), we showed that, in fact, hydrophobic patches were generated on the surface of the mutant protein. Those studies (3) also suggested that the binding site for Bis-ANS on P23T may coincide with the self-association site of the mutant and result in its lowered, retrograde solubility. Here we present new NMR-evidence to identify the Bis-ANS binding sites, and using independent NMR dynamics studies, also show that there is a measurable reduction in the flexibility of the peptide backbone near the hydrophobic patches. These two factors, namely the creation of hydrophobic patches and the lowered peptide backbone flexibility, taken together make a compelling argument that the hydrophobic patches may indeed facilitate the nucleation of aggregates of P23T which are held together by net hydrophobic interactions. Such aggregation would explain the retrograde solubility as well as the lens opacity in vivo. 1. Pande et al (2005) Biochemistry, 44, 2491-2500. 2. Pande et al (2009) Biochem. Biophys. Res. Commun., 382, 196-199. 3. Pande et al (2010) Biochemistry,49, 6122-6129. Supported by NIH grants GM085006 to AS and EY010535 to JP.

Investigating Protein Aggregation using Segmental Isotope Labeling and 2D IR Spectroscopy

Protein misfolding and aggregation are important processes in numerous human diseases, but the detailed structural changes that occur therein evade traditional crystallographic and NMR-based techniques. Rapid-scan 2D IR spectroscopy, combined with heavy isotope labeling, provides information on local secondary structure and coupling between IR chromophores, which can be used to study systems that aggregate in vitro on a timescale of minutes to hours. Recently, we have utilized single-position 1-13C=18O labels to develop a detailed kinetic model of the aggregation of synthetic hIAPP, a 37 AA peptide implicated in type II diabetes mellitus. Here we demonstrate a segmental, 13C labeling approach to the study the much larger human gammaD-crystallin, a 173 AA eye lens protein that aggregates via an unknown mechanism during cataract formation. Using bacterial expression and native chemical ligation, we have generated a gammaD-crystallin variant in which the highly similar N- and C-terminal domains are uniformly enriched in 12C and 13C, respectively. This results two well-resolved diagonal peaks in the 2D IR spectrum of the natively folded protein, with a separation of ∼40 cm−1. Aggregation under acidic conditions results in spectral changes consistent with amyloid fiber formation in the C-terminal domain and unfolding of the N-terminal domain, with evidence of interaction between the two based on the presence of cross-peaks. The structural model we propose, currently resolved at the level of individual domains, is nonetheless the most detailed available for a gammaD-crystallin aggregate to date. We anticipate that the method described will be broadly applicable to difficult systems including many aggregating, natively unstructured, or membrane-associated proteins.

Cataract-associated mutant E107A of human gammaD-crystallin shows increased attraction to alpha-crystallin and enhanced light scattering.

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.

Thermodynamic Studies on the Cataract-Associated Mutant, E107a, of Human Gamma-D Crystallin: Molecular Basis for Cataract Formation

The E107A mutation in human gammaD-crystallin (HGD) is associated with nuclear cataract. The concentration of crystallins in the lens is extremely high, approaching that within protein crystals. The composition in the lens nucleus, i.e. the central core, is such that the alpha- and gamma crystallin weight fractions are comparable. In earlier studies we showed that mutations in HGD dramatically compromised protein solubility, while maintaining the overall protein structure. In contrast, in the E107A mutation neither the structure (circular dichroism and tryptophan fluorescence emission) and stability (thermal denaturation), nor the solubility are affected significantly. However, as expected, the mutation raises the pI by ∼1 pH unit, i.e. from 7.4 to 8.4. The increase in pI suggested a change in the interaction of E107A with alpha crystallin, which is negatively charged at neutral pH. To determine changes in such interaction, we compared the liquid-liquid phase boundaries in binary mixtures containing either HGD or E107A, and alpha crystallin. Our preliminary studies show that while the phase-separation temperatures of mixtures in the two cases (i.e. HGD+alpha and E107A+alpha) remain comparable, the nature of the paired compositions of the two phases in equilibrium are distinct. In particular, the tie-line slopes are altered in the direction predicted to correspond to increased alpha-gamma attraction, on the basis of molecular dynamics simulation and thermodynamic perturbation theory (1). Thus, it appears that increased attractive interactions between the E107A mutant of HGD and alpha crystallin could destabilize the crystallin mixture in the lens nucleus, and lead to increased light scattering and cataract.(1) N. Dorsaz, G. M. Thurston, A. Stradner, P. Schurtenberger and G. Foffi, J. Phys. Chem. B 113:1693-1709 (2009)Supported by: NIH Grant EY 18249-01 (G.T.) and EY 10535 (J.P.)

Hydrophobic Core Mutations Associated with Cataract Development in Mice Destabilize Human γD-Crystallin

The human eye lens is composed of fiber cells packed with crystallins up to 450 mg/ml. Human γD-crystallin (HγD-Crys) is a monomeric, two-domain protein of the lens central nucleus. Both domains of this long lived protein have double Greek key β-sheet folds with well packed hydrophobic cores. Three mutations resulting in amino acid substitutions in the γ-crystallin buried cores (two in the N-terminal domain (N-td) and one in the C-terminal domain (C-td)) cause early onset cataract in mice, presumably an aggregated state of the mutant crystallins. It has not been possible to identify the aggregating precursor within lens tissues. To compare in vivo cataract-forming phenotypes with in vitro unfolding and aggregation of γ-crystallins, mouse mutant substitutions were introduced into HγD-Crys. The mutant proteins L5S, V75D, and I90F were expressed and purified from Escherichia coli. WT HγD-Crys unfolds in vitro through a three-state pathway, exhibiting an intermediate with the N-td unfolded and the C-td native-like. L5S and V75D in the N-td also displayed three-state unfolding transitions, with the first transition, unfolding of the N-td, shifted to significantly lower denaturant concentrations. I90F destabilized the C-td, shifting the overall unfolding transition to lower denaturant concentrations. During thermal denaturation, the mutant proteins exhibited lowered thermal stability compared with WT. Kinetic unfolding experiments showed that the N-tds of L5S and V75D unfolded faster than WT. I90F was globally destabilized and unfolded more rapidly. These results support models of cataract formation in which generation of partially unfolded species are precursors to the aggregated cataractous states responsible for light scattering.

Formation of Amyloid Fibrils In Vitro from Partially Unfolded Intermediates of Human γC-Crystallin

Mature-onset cataract results from the formation of light-scattering aggregates of lens crystallins. Although oxidative or mutational damage may be a prerequisite, little is known of the initiation or nucleation of these aggregated states. In mice carrying mutations in gamma-crystallin genes, a truncated form of gamma-crystallin formed intranuclear filamentous inclusions within lens fiber cells. Previous studies have shown that bovine crystallins and human gammaD-crystallin form amyloid fibrils under denaturing conditions in vitro. The amyloid fibril formation of human gammaC-crystallin (HgammaC-Crys) induced by low pH, together with characterization of a partially unfolded intermediate in the process were investigated. HgammaC-Crys was expressed and purified from Escherichia coli. Partially unfolded intermediates were detected by tryptophan fluorescence spectroscopy and UV resonance Raman spectroscopy. The aggregation into amyloid fibrils was monitored by solution turbidity and fluorescence assay. The morphology of aggregates was characterized using transmission electron microscopy (TEM). Secondary structure of the peptides in their fibrillar state was characterized using Fourier transform infrared spectroscopy (FTIR). The structure of HgammaC-Crys was perturbed at low pH. Partially unfolded intermediates were detected when solution pH was lowered to pH 3. At pH 3, HgammaC-Crys aggregated into amyloid fibrils. The kinetics and extent of the reaction was dependent on protein concentration, pH, and temperature. TEM images of aggregates revealed aggregation stages from short to long fibrils and from long fibrils to light-scattering fibril networks. FTIR spectroscopy confirmed the cross-beta character of the secondary structure of these fibrils. HgammaC-Crys formed amyloid fibrils on incubation at low pH via a partially unfolded intermediate. This process could contribute to the early stages of the formation of light-scattering species in the eye lens.

The Cataract-Associated R14C Mutant of Human γD-Crystallin Shows a Variety of Intermolecular Disulfide Cross-Links: A Raman Spectroscopic Study,

The Arg14 to Cys (R14C) mutation in the human gammaD-crystallin (HGD) gene has been associated with a juvenile-onset hereditary cataract. We showed previously [Pande, A., et al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 1993-1998] that rapid oxidation of Cys14 in the mutant leads to the formation of intermolecular, disulfide-cross-linked aggregates at physiological pH. Here we present a Raman spectroscopic analysis of R14C and HGD and show that R14C forms such aggregates even at pH 4.5. The lower pH enabled us to monitor the evolution of a variety of disulfide cross-links with distinct conformations around the CC-SS-CC dihedral angles. At least three cysteine residues are involved, forming protein-protein cross-links through disulfide-exchange reactions. From the pattern of the S-S and Trp Raman bands, we infer that Cys32 is likely to be involved in the cross-linking. The data suggest that protein precipitation in the mutant may not be the direct result of disulfide cross-linking, although such cross-linking is the initiating event. Thus, our Raman data not only enhance the understanding of the reactivity of Cys14 in the R14C mutant and the mechanism of opacity, but also shed light on the mechanism of oxidative degradation during long-term storage of thiol-containing pharmaceuticals.

NMR study of the cataract-linked P23T mutant of human γD-crystallin shows minor changes in hydrophobic patches that reflect its retrograde solubility

The Pro23 to Thr (P23T) mutation in human γD-crystallin (HGD) shows several cataract phenotypes. We found earlier [A. Pande, O. Annunziata, N. Asherie, O. Ogun, G.B. Benedek, J. Pande, Decrease in protein solubility and cataract formation caused by the Pro23 to Thr mutation in human gamma D-crystallin, Biochemistry 44 (2005) 2491–2500] that the mutation dramatically lowers the solubility of P23T but the overall protein fold is maintained. Recently we observed that solutions of P23T showed liquid–liquid phase transition behavior similar to that of HGD but the liquid–protein crystal phase transition was altered, suggesting an asymmetric distribution of “sticky” patches on the protein surface [J.J. McManus, A. Lomakin, O. Ogun, A. Pande, M. Basan, J. Pande, G.B. Benedek, Altered phase diagram due to a single point mutation in human gammaD-crystallin, Proc. Natl. Acad. Sci. USA 104 (2007) 16856–16861]. Here we present high-resolution NMR studies of HGD and P23T in which we have made nearly complete backbone assignments. The data provide a structural basis for explaining the retrograde solubility of P23T by (a) identifying possible “sticky” patches on the surface of P23T and (b) highlighting their asymmetric distribution.

The Structure of the Cataract-Causing P23T Mutant of Human γD-Crystallin Exhibits Distinctive Local Conformational and Dynamic Changes,

Crystallins are major proteins of the eye lens and essential for lens transparency. Mutations and aging of crystallins cause cataracts, the predominant cause of blindness in the world. In human γD-crystallin, the P23T mutant is associated with congenital cataracts. Until now, no atomic structural information has been available for this variant. Biophysical analyses of this mutant protein have revealed dramatically reduced solubility compared to that of the wild-type protein due to self-association into higher-molecular weight clusters and aggregates that retain a nativelike conformation within the monomers [Pande, A., et al. (2005) Biochemistry 44, 2491−2500]. To elucidate the structure and local conformation around the mutation site, we have determined the solution structure and characterized the protein’s dynamic behavior by NMR. Although the global structure is very similar to the X-ray structure of wild-type γD-crystallin, pivotal local conformational and dynamic differences are caused by the threonine substitution. In particular, in the P23T mutant, the imidazole ring of His22 switches from the predominant Nε2 tautomer in the wild-type protein to the Nδ1 tautomer, and an altered motional behavior of the associated region in the protein is observed. The data support structural changes that may initiate aggregation or polymerization by the mutant protein.

Visualization of In Situ Intracellular Aggregation of Two Cataract–Associated Human γ-Crystallin Mutants: Lose a Tail, Lose Transparency

To understand the molecular features underlying autosomal dominant congenital cataracts caused by the deletion mutations W156X in human gammaD-crystallin and W157X in human gammaC-crystallin. Normal and mutant cDNAs (with the enhanced green fluorescent protein [EGFP] tag in the front) were cloned into the pEGFP-C1 vector, transfected into various cell lines, and observed under a confocal microscope for EGFP fluorescence. Normal and W156X gammaD cDNAs were also cloned into the pET21a(+) vector, and the recombinant proteins were overexpressed in the BL-21(DE3)pLysS strain of Escherichia coli, purified, and isolated. The conformational features, structural stability, and solubility in aqueous solution of the mutant protein were compared with those of the wild type using spectroscopic methods. Comparative molecular modeling was performed to provide additional structural information. Transfection of the EGFP-tagged mutant cDNAs into several cell lines led to the visualization of aggregates, whereas that of wild-type cDNAs did not. Turning to the properties of the expressed proteins, the mutant molecules show remarkable reduction in solubility. They also seem to have a greater degree of surface hydrophobicity than the wild-type molecules, most likely accounting for self-aggregation. Molecular modeling studies support these features. The deletion of C-terminal 18 residues of human gammaC- and gammaD-crystallins exposes the side chains of several hydrophobic residues in the sequence to the solvent, causing the molecule to self-aggregate. This feature appears to be reflected in situ on the introduction of the mutants in human lens epithelial cells.

Folding and stability of the isolated Greek key domains of the long‐lived human lens proteins γD‐crystallin and γS‐crystallin

The transparency of the eye lens depends on the high solubility and stability of the lens crystallin proteins. The monomeric gamma-crystallins and oligomeric beta-crystallins have paired homologous double Greek key domains, presumably evolved through gene duplication and fusion. Prior investigation of the refolding of human gammaD-crystallin revealed that the C-terminal domain folds first and nucleates the folding of the N-terminal domain. This result suggested that the human N-terminal domain might not be able to fold on its own. We constructed and expressed polypeptide chains corresponding to the isolated N- and C-terminal domains of human gammaD-crystallin, as well as the isolated domains of human gammaS-crystallin. Both circular dichroism and fluorescence spectroscopy indicated that the isolated domains purified from Escherichia coli were folded into native-like monomers. After denaturation, the isolated domains refolded efficiently at pH 7 and 37 degrees C into native-like structures. The in vitro refolding of all four domains revealed two kinetic phases, identifying partially folded intermediates for the Greek key motifs. When subjected to thermal denaturation, the isolated N-terminal domains were less stable than the full-length proteins and less stable than the C-terminal domains, and this was confirmed in equilibrium unfolding/refolding experiments. The decrease in stability of the N-terminal domain of human gammaD-crystallin with respect to the complete protein indicated that the interdomain interface contributes of 4.2 kcal/mol to the overall stability of this very long-lived protein.

Mechanism of the Highly Efficient Quenching of Tryptophan Fluorescence in Human γD-Crystallin

Quenching of the fluorescence of buried tryptophans (Trps) is an important reporter of protein conformation. Human gammaD-crystallin (HgammaD-Crys) is a very stable eye lens protein that must remain soluble and folded throughout the human lifetime. Aggregation of non-native or covalently damaged HgammaD-Crys is associated with the prevalent eye disease mature-onset cataract. HgammaD-Crys has two homologous beta-sheet domains, each containing a pair of highly conserved buried tryptophans. The overall fluorescence of the Trps is quenched in the native state despite the absence of the metal ligands or cofactors. We report the results of detailed quantitative measurements of the fluorescence emission spectra and the quantum yields of numerous site-directed mutants of HgammaD-Crys. From fluorescence of triple Trp to Phe mutants, the homologous pair Trp68 and Trp156 were found to be extremely quenched, with quantum yields close to 0.01. The homologous pair Trp42 and Trp130 were moderately fluorescent, with quantum yields of 0.13 and 0.17, respectively. In an attempt to identify quenching and/or electrostatically perturbing residues, a set of 17 candidate amino acids around Trp68 and Trp156 were substituted with neutral or hydrophobic residues. None of these mutants showed significant changes in the fluorescence intensity compared to their own background. Hybrid quantum mechanical-molecular mechanical (QM-MM) simulations with the four different excited Trps as electron donors strongly indicate that electron transfer rates to the amide backbone of Trp68 and Trp156 are extremely fast relative to those for Trp42 and Trp130. This is in agreement with the quantum yields measured experimentally and consistent with the absence of a quenching side chain. Efficient electron transfer to the backbone is possible for Trp68 and Trp156 because of the net favorable location of several charged residues and the orientation of nearby waters, which collectively stabilize electron transfer electrostatically. The fluorescence emission spectra of single and double Trp to Phe mutants provide strong evidence for energy transfer from Trp42 to Trp68 in the N-terminal domain and from Trp130 to Trp156 in the C-terminal domain. The backbone conformation of tryptophans in HgammaD-Crys may have evolved in part to enable the lens to become a very effective UV filter, while the efficient quenching provides an in situ mechanism to protect the tryptophans of the crystallins from photochemical degradation.

Interdomain side‐chain interactions in human γD crystallin influencing folding and stability

Human gammaD crystallin (HgammaD-Crys) is a two domain, beta-sheet eye lens protein that must remain soluble throughout life for lens transparency. Single amino acid substitutions of HgammaD-Crys are associated with juvenile-onset cataracts. Features of the interface between the two domains conserved among gamma-crystallins are a central six-residue hydrophobic cluster, and two pairs of interacting residues flanking the cluster. In HgammaD-Crys these pairs are Gln54/Gln143 and Arg79/Met147. We previously reported contributions of the hydrophobic cluster residues to protein stability. In this study alanine substitutions of the flanking residue pairs were constructed and analyzed. Equilibrium unfolding/refolding experiments at 37 degrees C revealed a plateau in the unfolding/refolding transitions, suggesting population of a partially folded intermediate with a folded C-terminal domain (C-td) and unfolded N-terminal domain (N-td). The N-td was destabilized by substituting residues from both domains. In contrast, the C-td was not significantly affected by substitutions of either domain. Refolding rates of the N-td were significantly decreased for mutants of either domain. In contrast, refolding rates of the C-td were similar to wild type for mutants of either domain. Therefore, domain interface residues of the folded C-td probably nucleate refolding of the N-td. We suggest that these residues stabilize the native state by shielding the central hydrophobic cluster from solvent. Glutamine and methionine side chains are among the residues covalently damaged in aged and cataractous lenses. Such damage may generate partially unfolded, aggregation- prone conformations of HgammaD-Crys that could be significant in cataract.

Contributions of hydrophobic domain interface interactions to the folding and stability of human γD‐crystallin

Human gammaD-crystallin (HgammaD-Crys) is a monomeric eye lens protein composed of two highly homologous beta-sheet domains. The domains interact through interdomain side chain contacts forming two structurally distinct regions, a central hydrophobic cluster and peripheral residues. The hydrophobic cluster contains Met43, Phe56, and Ile81 from the N-terminal domain (N-td) and Val132, Leu145, and Val170 from the C-terminal domain (C-td). Equilibrium unfolding/refolding of wild-type HgammaD-Crys in guanidine hydrochloride (GuHCl) was best fit to a three-state model with transition midpoints of 2.2 and 2.8 M GuHCl. The two transitions likely corresponded to sequential unfolding/refolding of the N-td and the C-td. Previous kinetic experiments revealed that the C-td refolds more rapidly than the N-td. We constructed alanine substitutions of the hydrophobic interface residues to analyze their roles in folding and stability. After purification from E. coli, all mutant proteins adopted a native-like structure similar to wild type. The mutants F56A, I81A, V132A, and L145A had a destabilized N-td, causing greater population of the single folded domain intermediate. Compared to wild type, these mutants also had reduced rates for productive refolding of the N-td but not the C-td. These data suggest a refolding pathway where the domain interface residues of the refolded C-td act as a nucleating center for refolding of the N-td. Specificity of domain interface interactions is likely important for preventing incorrect associations in the high protein concentrations of the lens nucleus.