γD Crystallin Research Articles

Early Stage UV‐B Induced Molecular Modifications of Human Eye Lens γD‐Crystallin

Inside Front Cover: The human crystalline lens is composed of densely packed biopolymers that are constantly exposed to UV light throughout life and whose dysfunction leads to cataract. High-resolution protein NMR spectroscopy identified the early onset of molecular UV-B damage of aromatic amino acids in γD crystallin as part of the photoprotective capacity of the lens. This is reported by Jochen Balbach and co-workers in article number 2200526.

Elucidating the Effect of Point Mutations on Gamma B Crystallin Protein Interactions

Cataracts form when crystallin proteins in the eye lens undergo phase separation or aggregation, thereby failing to maintain lens transparency. Mutations in the amino acid sequence of the crystallins can alter the structure and/or chemical properties of the proteins, resulting in increased inter‐protein interactions, increased aggregation, or changes to the proteins’ phase diagram, all of which can enhance cataract formation. Here, we describe our work on γB crystallin, the bovine homologue to human γD crystallin, and two site‐directed mutants of γB crystallin with known links to congenital cataracts. Using nuclear magnetic resonance spectroscopy, we aim to determine how inter‐protein interactions of bovine γB crystallin are altered by point mutations so that we can gain a better understanding of the mechanism behind cataract formation. Results from preliminary experiments suggest that the inter‐protein interactions of γB crystallin are highly dependent on environmental conditions, such as temperature, pH, and protein concentration, and studies are underway to compare those behaviors to the behavior of mutant proteins under similar conditions.

UV radiation damage of human gamma D crystallin: role of tryptophans

Cataract is one of the main causes of blindness worldwide and is due to aggregation of eye lens proteins. Age, UV radiation, oxidation, metal ions, deamidation, and truncations are factor risks that cause protein damage and induce aggregation. Human γD (HgD) crystallin is one of the most abundant crystallins in the lens nucleus and its non-amyloid aggregation is

Double Domain Swapping in Human γC and γD Crystallin Drives Early Stages of Aggregation.

Human γD (HγD) and γC (HγC) are two-domain crystallin (Crys) proteins expressed in the nucleus of the eye lens. Structural perturbations in the protein often trigger aggregation, which eventually leads to cataract. To decipher the underlying molecular mechanism, it is important to characterize the partially unfolded conformations, which are aggregation-prone. Using a coarse grained protein model and molecular dynamics simulations, we studied the role of on-pathway folding intermediates in the early stages of aggregation. The multidimensional free energy surface revealed at least three different folding pathways with the population of partially structured intermediates. The two dominant pathways confirm sequential folding of the N-terminal [Ntd] and the C-terminal domains [Ctd], while the third, least favored, pathway involves intermediates where both the domains are partially folded. A native-like intermediate (I*), featuring the folded domains and disrupted interdomain contacts, gets populated in all three pathways. I* forms domain swapped dimers by swapping the entire Ntds and Ctds with other monomers. Population of such oligomers can explain the increased resistance to unfolding resulting in hysteresis observed in the folding experiments of HγD Crys. An ensemble of double domain swapped dimers are also formed during refolding, where intermediates consisting of partially folded Ntds and Ctds swap secondary structures with other monomers. The double domain swapping model presented in our study provides structural insights into the early events of aggregation in Crys proteins and identifies the key secondary structural swapping elements, where introducing mutations will aid in regulating the overall aggregation propensity.

Mercury-induced aggregation of human lens γ-crystallins reveals a potential role in cataract disease.

Cataract disease results from non-amyloid aggregation of eye lens proteins and is the leading cause of blindness in the world. A variety of studies have implicated both essential and xenobiotic metals as potential etiological agents in cataract disease. Essential metal ions, such as copper and zinc, are known to induce the aggregation in vitro of human γD crystallin, one of the more abundant γ-crystallins in the core of the lens. In this study, we expand the investigation of metal-crystallin interactions to heavy metal ions, such as divalent lead, cadmium and mercury. The impact of these metal ions in the non-amyloid aggregation, protein folding and thermal stability of three homologous human lens γ-crystallins has been evaluated using turbidity assays, electron microscopy, electronic absorption and circular dichroism spectroscopies. Our results show that Hg(II) ions can induce the non-amyloid aggregation of human γC and γS crystallins, but not γD crystallin. The mechanism of Hg-induced aggregation involves direct metal-protein interactions, loss of thermal stability, partial unfolding of the N-terminal domain of these proteins, and formation of disulfide-bridged dimers. Putative Hg(II) binding sites in γ-crystallins involved in metal-induced aggregation are discussed. This study reveals that mercury ions can induce the aggregation of human lens proteins, uncovering a potential role of this heavy metal ion in the bioinorganic chemistry of cataract disease.

A molecular dynamics approach to explore the structural characterization of cataract causing mutation R58H on human γD crystallin.

The crystallins are a family of monomeric proteins present in the mammalian lens and mutations in these proteins cause various forms of cataracts. The aim of our current study is to emphasize the structural characterization of aggregation propensity of mutation R58H on γD crystallin using molecular dynamics (MD) approach. MD result revealed that difference in the sequence level display a wide variation in the backbone atomic position, and thus exhibits rigid conformational dynamics. Changes in the flexibility of residues favoured to increase the number of intra-molecular hydrogen bonds in mutant R58H. Moreover, notable changes in the hydrogen bonding interaction resulted to cause the misfolding of mutant R58H by introducing α-helix. Principal component analysis (PCA) result suggested that mutant R58H showed unusual conformational dynamics along the two principal components when compared to the wild-type (WT)-γD crystallin. In a nutshell, the increased surface hydrophobicity could be the cause of self-aggregation of mutant R58H leading to aculeiform cataract.

WT Human γD Crystallin Promotes Aggregation of its Oxidation-Mimicking Mutants

Non-native protein conformers generated by mutation or chemical damage template aggregation of wild-type, undamaged polypeptides in diseases ranging from amyotrophic lateral sclerosis to cancer. We tested for such interactions in the natively monomeric human eye lens protein γD-crystallin, whose aggregation leads to cataract disease. The oxidation-mimicking W42Q mutant of γD-crystallin formed non-native polymers starting from a native-like state under physiological conditions in vitro. Aggregation occurred in the temperature range 35-45 oC, in which the mutant protein began to lose the native conformation of its N-terminal domain. Surprisingly, it was wild-type γD-crystallin that promoted W42Q polymerization in a catalytic manner, even at mutant concentrations too low for homogeneous nucleation to occur. WT also downshifted the temperature range of W42Q aggregation. Aggregation required formation of a non-native internal disulfide bond in the mutant protein, which may result from a conformational rearrangement catalyzed by WT binding. Similar results were obtained for the W42R mutant, which causes congenital cataract. Monte-Carlo simulations with a knowledge-based statistical potential showed melting of specific residue contacts as a result of hydrophilic Trp substitutions. WT and W42Q populated similar early unfolding intermediates, which were able to domain-swap in two-chain simulations. The WT/W42Q heterodimer was longer-lived in silico than the W42Q/W42Q homodimer, suggesting a possible mechanism for WT-catalyzed aggregation of mutant or modified protein as oxidative damage accumulates during the course of aging.

Elucidating the Coordination Features Associated to Copper and Zinc Induced Aggregation of Human γ-D Crystallin

Cataract is the leading cause of blindness in the world. The monomeric all -sheet Human γD (HγD) crystallin is an abundant crystallin in the core of the lens and its non-amyloid aggregation is associated with cataracts. A variety of experimental and etiological studies implicate metals as a potential etiological agent for cataract. Copper and zinc concentrations in the cataractous lenses are increased significantly, as compared to normal lenses. Micromolar concentrations of Cu(II) and Zn(II) ions specifically induce the aggregation of HγD crystallin. The mechanism for metal-induced aggregation likely involves interaction of the metal ions with the protein, leading to partial unfolding and formation of high molecular weight light scattering aggregates. In this work, we study the coordination features associated to the metal-induced aggregation of HγD crystallin and probe it by site-directed mutagenesis. Several His and Cys residues have been identified as putative metal anchoring sites. The effects of mutations at these residues in the copper- and zinc- induced aggregation of HγD crystallin have been evaluated. A specific interaction of Zn(II) ions with the N-terminal domain that is key for zinc-induced aggregation has been identified. On the other hand, circular dichroism and electron paramagnetic resonance data show that Cu(II) ions can bind at more than one site in the protein. These results will help elucidate the mechanism by which copper and zinc ions induce the aggregation of HγD crystallin, as it relates to cataracts disease. This research has been supported by: MIT-Seed Funds, Conacyt (#221134), NIH EY015834, and Fulbright-Garcia Robles fellowship to L.Q.

Copper and Zinc Ions Specifically Promote Nonamyloid Aggregation of the Highly Stable Human γ-D Crystallin

Cataract is the leading cause of blindness in the world. It results from aggregation of eye lens proteins into high-molecular-weight complexes, causing light scattering and lens opacity. Copper and zinc concentrations in cataractous lens are increased significantly relative to a healthy lens, and a variety of experimental and epidemiological studies implicate metals as potential etiological agents for cataract. The natively monomeric, β-sheet rich human γD (HγD) crystallin is one of the more abundant proteins in the core of the lens. It is also one of the most thermodynamically stable proteins in the human body. Surprisingly, we found that both Cu(II) and Zn(II) ions induced rapid, nonamyloid aggregation of HγD, forming high-molecular-weight light-scattering aggregates. Unlike Zn(II), Cu(II) also substantially decreased the thermal stability of HγD and promoted the formation of disulfide-bridged dimers, suggesting distinct aggregation mechanisms. In both cases, however, metal-induced aggregation depended strongly on temperature and was suppressed by the human lens chaperone αB-crystallin (HαB), implicating partially folded intermediates in the aggregation process. Consistently, distinct site-specific interactions of Cu(II) and Zn(II) ions with the protein and conformational changes in specific hinge regions were identified by nuclear magnetic resonance. This study provides insights into the mechanisms of metal-induced aggregation of one of the more stable proteins in the human body, and it reveals a novel and unexplored bioinorganic facet of cataract disease.

Copper and Zinc Binding Specifically Induce the Aggregation of Human γ-D Crystallin

Cataract is the leading cause of blindness in the world, and it is projected to affect 20-30 million people in 2020. Cataracts are formed upon aggregation of lens proteins into high molecular weight complexes, causing light scattering and lens opacity (1). A variety of experimental and etiological studies implicate metals as a potential etiological agent for cataract. Copper and zinc concentrations in the cataractous lenses are increased significantly, as compared to normal lenses (2). The monomeric all β-sheet Human γD (HγD) crystallin is one of the abundant crystallins in the core of the lens and its non-amyloid aggregation is associated with cataracts.1 We find that micromolar concentrations of Cu(II) and Zn(II) ions exert distinct and specific effects on the conformation of HγD, suggesting site-specific interactions. Circular dichroism studies show that Cu(II) induces loss of secondary structure and stability of HγD. Spectroscopic studies demonstrate that Cu(II) ions can bind at more than one site in the protein. Metal binding to the monomer leads to formation of high molecular weight light scattering aggregates. These metal-induced effects occur at micromolar concentrations of metal ion and protein, starting at metal:protein ratios of 1:1. Interestingly, the lens chaperone αB crystallin protected HγD from metal-induced aggregation. Metal-induced aggregation could be a physiologically relevant phenomenon; understanding its mechanism will help elucidate the role of metal ions in the aggregation of human crystallins and their potential involvement in the development of cataracts. This research has been supported by: MIT-Seed Funds, Conacyt (grant # 221134 and fellowship to J.A.D.-C.), NIH EY015834, AMC-FUMEC and Fulbright-Garcia Robles fellowships to L.Q.1. Moureau, L.K.; King A.J. Trends Mol. Med., 2012, 18, 273-2822. Curtis, E.D. Exp. Eye Res., 1983, 37, 639-647

Real Time NMR Folding Study of the Human Gamma D Crystallin in the Presence of Metal Ions

Cataract is a leading cause of blindness worldwide, and is due to aggregation of eye lens proteins. Age, UV radiation, oxidation, metal ions, deamidation, and truncations may be cause covalent protein damage that induces aggregation. In particular, metal ions have been implicated as a potential etiological agent for cataract. Human γD (HγD) crystallin is one of the most abundant crystallins in the lens nucleus and its non-amyloid aggregation is associated with cataracts. HγD is a highly stable protein with two homologous domains (N-terminal and C-terminal), each containing two Greek key motifs forming a β-sandwich of eight intercalated β-strands. Domain swapping from a perturbed conformation has been proposed as the mechanism of aggregation. Micromolar concentrations of Cu(II) and Zn(II) ions specifically induces the aggregation of this stable protein. In this work, NMR (H-N-HSQC) experiments were performed to study the interaction of Cu(II) and Zn(II) ions with HγD and identify possible specific metal binding sites in the protein. Moreover, we have characterized the unfolding of HγD in the presence of these ions with atomic resolution using real-time NMR spectroscopy. These studies provide further insight into the metal ion coordination properties of HγD, and the role of Cu(II) and Zn(II) in its non-amyloid aggregation. This research has been supported by: MIT-Seed Funds and NIH EY015834.

Conformational stability as a design target to control protein aggregation.

Non-native protein aggregation is a prevalent problem occurring in many biotechnological manufacturing processes and can compromise the biological activity of the target molecule or induce an undesired immune response. Additionally, some non-native aggregation mechanisms lead to amyloid fibril formation, which can be associated with debilitating diseases. For natively folded proteins, partial or complete unfolding is often required to populate aggregation-prone conformational states, and therefore one proposed strategy to mitigate aggregation is to increase the free energy for unfolding (ΔGunf) prior to aggregation. A computational design approach was tested using human γD crystallin (γD-crys) as a model multi-domain protein. Two mutational strategies were tested for their ability to reduce/increase aggregation rates by increasing/decreasing ΔGunf: stabilizing the less stable domain and stabilizing the domain-domain interface. The computational protein design algorithm, RosettaDesign, was implemented to identify point variants. The results showed that although the predicted free energies were only weakly correlated with the experimental ΔGunf values, increased/decreased aggregation rates for γD-crys correlated reasonably well with decreases/increases in experimental ΔGunf, illustrating improved conformational stability as a possible design target to mitigate aggregation. However, the results also illustrate that conformational stability is not the sole design factor controlling aggregation rates of natively folded proteins.

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.

Is protein methylation in the human lens a result of non-enzymatic methylation by S-adenosylmethionine?

Since crystallins in the human lens do not turnover, they are susceptible to modification by reactive molecules over time. Methylation is a major post-translational lens modification, however the source of the methyl group is not known and the extent of modification across all crystallins has yet to be determined. Sites of methylation in human lens proteins were determined using HPLC/mass spectrometry following digestion with trypsin. The overall extent of protein methylation increased with age, and there was little difference in the extent of modification between soluble and insoluble crystallins. Several different cysteine and histidine residues in crystallins from adult lenses were found to be methylated with one cysteine (Cys 110 in γD crystallin) at a level approaching 70%, however, methylation of crystallins was not detected in fetal or newborn lenses. S-adenosylmethionine (SAM) was quantified at significant (10–50 μM) levels in lenses, and in model experiments SAM reacted readily with N-α-tBoc-cysteine and N-α-tBoc-histidine, as well as βA3-crystallin. The pattern of lens protein methylation seen in the human lens was consistent with non-enzymatic alkylation. The in vitro data shows that SAM can act directly to methylate lens proteins and SAM was present in significant concentrations in human lens. Thus, non-enzymatic methylation of crystallins by SAM offers a possible explanation for this major human lens modification.

Inhibition of unfolding and aggregation of lens protein human gamma D crystallin by sodium citrate

Cataract affects 1 in 6 Americans over the age of 40, and represents a global health problem. Mature onset cataract is associated with the aggregation of partially unfolded or damaged proteins in the lens, which accumulate as an individual ages. Currently, surgery is the primary effective treatment for cataract. As an alternative preventive approach, small molecules have been suggested as potential therapeutic agents. In this work, we study the effect of sodium citrate on the stability of Human γD Crystallin (HγD-Crys), a structural protein of the eye lens, and two cataract-related mutants, L5S HγD-Crys and I90F HγD-Crys. In equilibrium unfolding–refolding studies, the presence of 250 mM sodium citrate increased the transition midpoint of the N-terminal domain (N-td) of WT HγD-Crys and L5S HγD-Crys by 0.3 M GuHCl, the C-terminal domain (C-td) by 0.6 M GuHCl, and the single transition of I90F HγD-Crys by 0.4 M GuHCl. In kinetic unfolding reactions, sodium citrate stabilization effect was observed only for the mutant I90F HγD-Crys. In the presence of citrate, a kinetic unfolding intermediate of I90F HγD-Crys was observed, which was not populated in the absence of citrate. The rates of aggregation were measured using solution turbidity. Sodium citrate demonstrated negligible effect on rate of aggregation of WT HγD-Crys, but considerably slowed the rate of aggregation of both L5S HγD-Crys and I90F HγD-Crys. The presence of sodium citrate dramatically slowed refolding of WT HγD-Crys and I90F HγD-Crys, but had a significantly smaller effect on the refolding of L5S HγD-Crys. The differential stabilizing effect of sodium citrate suggests that the ion is binding to a partially unfolded conformation of the C-td, but a solution-based Hofmeister effect cannot be eliminated as a possible explanation for the effects observed. These results indicate that assessment of potential anti-cataract agents needs to include effects on the unfolding and aggregation pathways, as well as the native state.

Implementation of a Project-Based Molecular Biology Laboratory Emphasizing Protein Structure–Function Relationships in a Large Introductory Biology Laboratory Course

In introductory laboratory courses, many universities are turning from traditional laboratories with predictable outcomes to inquiry-inspired, project-based laboratory curricula. In these labs, students are allowed to design at least some portion of their own experiment and interpret new, undiscovered data. We have redesigned the introductory biology laboratory course at Brandeis University into a semester-long project-based laboratory that emphasizes concepts and contains an element of scientific inquiry. In this laboratory, students perform a site-directed mutagenesis experiment on the gene encoding human γD crystallin, a human eye lens protein implicated in cataracts, and assess the stability of their newly created protein with respect to wild-type crystallin. This laboratory utilizes basic techniques in molecular biology to emphasize the importance of connections between DNA and protein. This project lab has helped engage students in their own learning, has improved students’ skills in critical thinking and analysis, and has promoted interest in basic research in biology.

Computational Design and Biophysical Characterization of Aggregation-Resistant Point Mutations for γD Crystallin Illustrate a Balance of Conformational Stability and Intrinsic Aggregation Propensity

γD crystallin is a natively monomeric eye-lens protein that is associated with hereditary juvenile cataract formation. It is an attractive model system as a multidomain Greek-key protein that aggregates through partially folded intermediates. Point mutations M69Q and S130P were used to test (1) whether the protein-design algorithm RosettaDesign would successfully predict mutants that are resistant to aggregation when combined with informatic sequence-based predictors of peptide aggregation propensity and (2) how the mutations affected relative unfolding free energies (ΔΔG(un)) and intrinsic aggregation propensity (IAP). M69Q was predicted to have ΔΔG(un) ≫ 0, without significantly affecting IAP. S130P was predicted to have ΔΔG(un) ∼ 0 but with reduced IAP. The stability, conformation, and aggregation kinetics in acidic solution were experimentally characterized and compared for the variants and wild-type (WT) protein using circular dichroism and intrinsic fluorescence spectroscopy, calorimetric and chemical unfolding, thioflavin-T binding, chromatography, static laser light scattering, and kinetic modeling. Monomer secondary and tertiary structures of both variants were indistinguishable from WT, while ΔΔG(un) > 0 for M69Q and ΔΔG(un) < 0 for S130P. Surprisingly, despite being the least conformationally stable, S130P was the most resistant to aggregation, indicating a significant decrease of its IAP compared to WT and M69Q.

The group II chaperonin Mm‐Cpn binds and refolds human γD crystallin

Chaperonins assist in the folding of nascent and misfolded proteins, though the mechanism of folding within the lumen of the chaperonin remains poorly understood. The archeal chaperonin from Methanococcus marapaludis, Mm-Cpn, shares the eightfold double barrel structure with other group II chaperonins, including the eukaryotic TRiC/CCT, required for actin and tubulin folding. However, Mm-Cpn is composed of a single species subunit, similar to group I chaperonin GroEL, rather than the eight subunit species needed for TRiC/CCT. Features of the β-sheet fold have been identified as sites of recognition by group II chaperonins. The crystallins, the major components of the vertebrate eye lens, are β-sheet proteins with two homologous Greek key domains. During refolding in vitro a partially folded intermediate is populated, and partitions between productive folding and off-pathway aggregation. We report here that in the presence of physiological concentrations of ATP, Mm-Cpn suppressed the aggregation of HγD-Crys by binding the partially folded intermediate. The complex was sufficiently stable to permit recovery by size exclusion chromatography. In the presence of ATP, Mm-Cpn promoted the refolding of the HγD-Crys intermediates to the native state. The ability of Mm-Cpn to bind and refold a human β-sheet protein suggests that Mm-Cpn may be useful as a simplified model for the substrate recognition mechanism of TRiC/CCT.

Citrate Binding Stabilizes Human Gamma-Crystallin to Slow Unfolding and Inhibit Aggregation

Recent studies have demonstrated that small molecules can bind destabilized or aggregation prone proteins to prevent unfolding and aggregation. Cataract, the leading cause of blindness worldwide, is caused by aggregation of proteins in the eye lens. The most abundant proteins in the eye lens belongs to the βγ-Crystallin superfamily, which accounts for 90% of lens protein composition. These proteins are synthesized in utero and must remain stable and soluble throughout life. Damage, or deleterious post-translational modification can destabilize these proteins and induce aggregation-prone conformations. Sodium citrate has been shown to prevent unfolding and aggregation of alpha-antitrypsin by stabilizing secondary structure. In this study, we demonstrate the effects of citrate binding on both wild-type and disease models of Human γD Crystallin. Equilibrium unfolding-refolding experiments show an increase in the ΔG of unfolding with increasing concentrations of sodium citrate, while kinetic experiments show that sodium citrate slows the rate of unfolding in denaturant. UV resonance Raman spectroscopy has been used to examine Trp residues in the protein and monitor vibrational modes as a function of temperature. Preliminary results indicate a resistance to unfolding in the presence of citrate. The effect does not appear to be due to metal ion chelation, and may reflect direct binding to the crystallins, as with anti-trypsin.

Stability and aggregation differences in two lens γ‐crystallins, proteins implicated in cataract

The transparency of the eye lens depends on the high solubility and stability of the crystallin proteins. Aggregation of partially unfolded or covalently damaged forms results in mature-onset cataracts. Thus, the biochemical basis of the extremely high stability of the crystallins, and the nature of the misfolded, modified, or aggregated states, are important in understanding the etiology of cataract. We analyzed the biophysical properties of human γD crystallin (γD-Crys) and human γS crystallin (γS-Crys), two of the most abundant proteins in the lens. γD-Crys is synthesized in utero and must remain soluble throughout life in the differentiated enucleated lens fiber cells. In contrast, γS-Crys is expressed postpartum and throughout life in the fiber cells. Comparison of these proteins’s conformational stability indicated that γD-Crys is more stable than γS-Crys. In addition, we analyzed the unfolding kinetic rates of γD-Crys and γS-Crys. γD-Crys was highly stable kinetically with an extrapolated half-life of 19 years while γS-Crys had a 7.5 hour half-life for the first kinetic unfolding step. Although structurally similar, these crystallins exhibit different conformational and kinetic stability with γD-Crys more stable than γS-Crys. Given the physiological differences of these crystallins in the lens, these results demonstrate the high stability requirement for long-lived crystallins in the lens.