The importance of the fourth Greek key motif of human γD-crystallin in maintaining lens transparency-the tale told by the tail.

Purpose: Congenital cataract affects 1-15 per 10,000 newborns worldwide, and 20,000-40,000 children are born every year with developmental bilateral cataracts. Mutations in the crystallin genes are known to cause congenital cataracts. Crystallins, proteins present in the eye lens, are made up of four Greek key motifs separated into two domains. Greek key motifs play an important role in compact folding to provide the necessary refractive index and transparency. The present study was designed to understand the importance of the fourth Greek key motif in maintaining lens transparency by choosing a naturally reported Y134X mutant human γD- crystallin in a Danish infant and its relationship to lens opacification and cataract. Methods: Human γD-crystallin complementary DNA (cDNA) was cloned into the pET-21a vector, and the Y134X mutant clone was generated by site-directed mutagenesis. Wild-type and mutant proteins were overexpressed in the BL21 DE3 pLysS cells of E. coli. Wild-type protein was purified from the soluble fraction using the ion exchange and gel filtration chromatography methods. Mutant protein was predominantly found in insoluble fraction and purified from inclusion bodies. The structure, stability, aggregational, and amyloid fibril formation properties of the mutant were compared to those of the wild type using the fluorescence and circular dichroism spectroscopy methods. Results: Loss of the fourth Greek key motif in human γD-crystallin affects the backbone conformation, alters the tryptophan micro-environment, and exposes a nonpolar hydrophobic core to the surface. Mutant is less stable and opens its Greek key motifs earlier with a concentration midpoint (CM) of unfolding curve of 1.5 M compared to the wild type human γD-crystallin (CM: 2.5 M). Mutant is capable of forming self-aggregates immediately in response to heating at 48.6 °C. Conclusions: Loss of 39 amino acids in the fourth Greek key motif of human γD-crystallin affects the secondary and tertiary structures and exposes the hydrophobic residues to the solvent. These changes make the molecule less stable, resulting in the formation of light-scattering particles, which explains the importance of the fourth Greek key in the underlying mechanism of opacification and cataract.

Molecular Insights into the Inhibitory Role of α-Crystallin against γD-Crystallin Aggregation.

Cataracts, a major cause of global blindness, contribute significantly to the overall prevalence of blindness. The opacification of the lens, resulting in cataract formation, primarily occurs due to the aggregation of crystallin proteins within the eye lens. Despite the high concentration of these crystallins, they remarkably maintain the lens transparency and refractive index. α-Crystallins (α-crys), acting as chaperones, play a crucial role in preventing crystallin aggregation, although the exact molecular mechanism remains uncertain. In this study, we employed a combination of molecular docking, all-atom molecular dynamics simulations, and advanced free energy calculations to investigate the interaction between γD-crystallin (γD-crys), a major structural protein of the eye lens, and α-crystallin proteins. Our findings demonstrate that α-crys exhibits an enhanced affinity for the NTD2 and CTD4 regions of γD-crys. The NTD2 and CTD4 regions form the interface between the N-terminal domain (NTD) and the C-terminal domain (CTD) of the γD-crys protein. By binding to the interface region between the NTD and CTD of the protein, α-crys effectively inhibits the formation of domain-swapped aggregates and mitigates protein aggregation. Analysis of the Markov state models using molecular dynamics trajectories confirms that minimum free energy conformations correspond to the binding of the α-crystallin domain (ACD) of α-crys to NTD2 and CTD4 that form the interdomain interface.

Novel CRYGC Mutation in Conserved Ultraviolet-Protective Tryptophan (p.Trp131Arg) Is Linked to Autosomal Dominant Congenital Cataract.

Congenital cataract (CC), the most prevalent cause of childhood blindness and amblyopia, necessitates prompt and precise genetic diagnosis. The objective of this study is to identify the underlying genetic cause in a Swiss patient with isolated CC. Whole exome sequencing (WES) and copy number variation (CNV) analysis were conducted for variant identification in a patient born with a total binocular CC without a family history of CC. Sanger Sequencing was used to confirm the variant and segregation analysis was used to screen the non-affected parents. The first de novo missense mutation at c.391T>C was identified in exon 3 of CRYGC on chromosome 2 causing the substitution of a highly conserved Tryptophan to an Arginine located at p.Trp131Arg. Previous studies exhibit significant changes in the tertiary structure of the crystallin family in the following variant locus, making CRYGC prone to aggregation aggravated by photodamage resulting in cataract. The variant can be classified as pathogenic according to the American College of Medical Genetics and Genomics (ACMG) criteria (PP3 + PM1 + PM2 + PS2; scoring 10 points). The identification of this novel variant expands the existing knowledge on the range of variants found in the CRYGC gene and contributes to a better comprehension of cataract heterogeneity.

Cataract-causing mutations S78F and S78P of γD-crystallin decrease protein conformational stability and drive aggregation.

Congenital cataract is the leading cause of childhood blindness, which primarily results from genetic factors. γD-crystallin is the most abundant γ-crystallin and is essential for maintaining lens transparency and refractivity. Numerous mutations in γD-crystallin have been reported with unclear pathogenic mechanism. Two different cataract-causing mutations Ser78Phe and Ser78Pro in γD-crystallin were previously identified at the same conserved Ser78 residue. In this work, firstly, we purified the mutants and characterized for the structural change using fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and size-exclusion chromatography (SEC). Both mutants were prone to form insoluble precipitates when expressed in Escherichia coli strain BL21 (DE3) cells. Compared with wild-type (WT), both mutations caused structural disruption, increased hydrophobic exposure, decreased solubility, and reduced thermal stability. Next, we investigated the aggregation of the mutants at the cellular level. Overexpression the mutants in HLE-B3 and HEK 293T cells could induce aggresome formations. The environmental stresses (including heat, ultraviolet irradiation and oxidative stress) promoted the formation of aggregates. Moreover, the intracellular S78F and S78P aggregates could be reversed by lanosterol. Molecular dynamic simulation indicated that both mutations disrupted the structural integrity of Greek-key motif 2. Hence, our results reveal the vital role of conserved Ser78 in maintaining the structural stability, which can offer new insights into the mechanism of cataract formation.

Human γS-Crystallin Mutation F10_Y11delinsLN in the First Greek Key Pair Destabilizes and Impairs Tight Packing Causing Cortical Lamellar Cataract.

Aromatic residues forming tyrosine corners within Greek key motifs are critical for the folding, stability, and order of ßγ-crystallins and thus lens transparency. To delineate how a double amino acid substitution in an N-terminal-domain tyrosine corner of the CRYGS mutant p.F10_Y11delinsLN causes juvenile autosomal dominant cortical lamellar cataracts, human γS-crystallin c-DNA was cloned into pET-20b (+) and a p.F10_Y11delinsLN mutant was generated via site-directed mutagenesis, overexpressed, and purified using ion-exchange and size-exclusion chromatography. Structure, stability, and aggregation properties in solution under thermal and chemical stress were determined using spectrofluorimetry and circular dichroism. In benign conditions, the p.F10_Y11delinsLN mutation does not affect the protein backbone but alters its tryptophan microenvironment slightly. The mutant is less stable to thermal and GuHCl-induced stress, undergoing a two-state transition with a midpoint of 60.4 °C (wild type 73.1 °C) under thermal stress and exhibiting a three-state transition with midpoints of 1.25 and 2.59 M GuHCl (wild type: two-state transition with Cm = 2.72 M GuHCl). The mutant self-aggregates upon heating at 60 °C, which is inhibited by α-crystallin and reducing agents. Thus, the F10_Y11delinsLN mutation in human γS-crystallin impairs the protein's tryptophan microenvironment, weakening its stability under thermal and chemical stress, resulting in self-aggregation, lens opacification, and cataract.

Atomistic modeling of liquid-liquid phase equilibrium explains dependence of critical temperature on γ-crystallin sequence.

Liquid-liquid phase separation of protein solutions has regained heightened attention for its biological importance and pathogenic relevance. Coarse-grained models are limited when explaining residue-level effects on phase equilibrium. Here we report phase diagrams for γ-crystallins using atomistic modeling. The calculations were made possible by combining our FMAP method for computing chemical potentials and Brownian dynamics simulations for configurational sampling of dense protein solutions, yielding the binodal and critic temperature (Tc). We obtain a higher Tc for a known high-Tc γ-crystallin, γF, than for a low-Tc paralog, γB. The difference in Tc is corroborated by a gap in second virial coefficient. Decomposition of inter-protein interactions reveals one amino-acid substitution between γB and γF, from Ser to Trp at position 130, as the major contributor to the difference in Tc. This type of analysis enables us to link phase equilibrium to amino-acid sequence and to design mutations for altering phase equilibrium.

Identification of the Most Impactful Asparagine Residues for γS-Crystallin Aggregation by Deamidation.

Crystallin aggregation in the eye lens is involved in the pathogenesis of cataracts. The aggregation is considered to be promoted by non-enzymatic post-translational modifications, such as the deamidation and stereoinversion of amino acid residues. Although in a previous study, the deamidated asparagine residues were detected in γS-crystallin in vivo, it is unclear which deamidated residues have the most impact on the aggregation under physiological conditions. In this study, we investigated the deamidation impacts of all Asn residues in γS-crystallin for the structural and aggregation properties utilizing deamidation mimetic mutants (N14D, N37D, N53D, N76D, and N143D). The structural impacts were investigated using circular dichroism analysis and molecular dynamics simulations, and the aggregation properties were analyzed by gel filtration chromatography and spectrophotometric methods. No significant structural impacts of all mutations were detected. However, the N37D mutation decreased thermal stability and changed some intermolecular hydrogen-bond formations. Aggregation analysis indicated that the superiority of the aggregation rate in each mutant varied with temperature. Deamidation at any Asn residues promoted γS-crystallin aggregation, and the deamidation at Asn37, Asn53, and Asn76 were suggested to be the most impactful in the formation of insoluble aggregations.

The Y46D Mutation Destabilizes Dense Packing of the Second Greek Key Pair of Human γC-Crystallin Causing Congenital Nuclear Cataracts.

The γ-crystallins are highly expressed structural lens proteins comprising four Greek key motifs arranged in two domains. Their globular structure and short-range spatial ordering are essential for lens transparency. Aromatic residues play a vital role in stabilizing Greek key folds by forming Greek key or non-Greek key pairs or tyrosine corners. We investigated the effects of the cataractogenic Y46D mutation in the second Greek key pair (Y46-Y51) of human γC-crystallin on its stability and aggregation. Wild-type and Y46D mutant human γC-crystallin were overexpressed in E. coli BL-21(DE3) PLysS cells, purified using ion-exchange and size-exclusion chromatography, and analyzed by fluorescence spectroscopy and circular dichroism spectroscopy. The Y46D mutation does not affect the γC-crystallin backbone conformation under benign conditions but alters the tryptophan microenvironment, exposing hydrophobic residues to the surface. The Y46D mutant undergoes a three-state transition under thermal stress with midpoints of 54.6 and 67.7 °C while the wild type shows a two-state transition with a midpoint of 77.6 °C. The Y46D mutant also shows a three-state transition under GuHCl stress with Cm values of 0.9 and 2.1 M while the wild type shows a two-state transition with a Cm of 2.4 M GuHCl. Mutant but not wild-type γC-crystallin forms light scattering particles upon heating at 65 °C. Overall, the Y46D CRYGS mutation leaves the protein fold intact under benign conditions but destabilizes the molecule by altering the tryptophan microenvironment and exposing hydrophobic residues to its surface, thus increasing its susceptibility to thermal and chemical stress with resultant self-aggregation, light scattering, and cataract.

Insights into the solubility of γ D-crystallin from multiscale atomistic simulations.

The molecular basis underlying the rich phase behavior of globular proteins remains poorly understood. We use atomistic multiscale molecular simulations to model the solution-state conformational dynamics and interprotein interactions of γ D-crystallin and its P23T-R36S mutant, which drastically limits the protein solubility, at both infinite dilution and at a concentration where the mutant fluid phase and crystalline phase coexist. We find that while the mutant conserves the protein fold, changes to the surface exposure of residues in the neighborhood of residue-36 enhance protein-protein interactions and develop specific protein-protein contacts found in the protein crystal lattice.

Copper Reductase Activity and Free Radical Chemistry by Cataract-Associated Human Lens γ-Crystallins.

Cataracts are caused by high-molecular-weight aggregates of human eye lens proteins that scatter light, causing lens opacity. Metal ions have emerged as important potential players in the etiology of cataract disease, as human lens γ-crystallins are susceptible to metal-induced aggregation. Here, the interaction of Cu2+ ions with γD-, γC-, and γS-crystallins, the three most abundant γ-crystallins in the lens, has been evaluated. Cu2+ ions induced non-amyloid aggregation in all three proteins. Solution turbidimetry, sodium dodecyl sulfate poly(acrylamide) gel electrophoresis (SDS-PAGE), circular dichroism, and differential scanning calorimetry showed that the mechanism for Cu-induced aggregation involves: (i) loss of ß-sheet structure in the N-terminal domain; (ii) decreased thermal and kinetic stability; (iii) formation of metal-bridged species; and (iv) formation of disulfide-bridged dimers. Isothermal titration calorimetry (ITC) revealed distinct Cu2+ binding affinities in the γ-crystallins. Electron paramagnetic resonance (EPR) revealed two distinct Cu2+ binding sites in each protein. Spin quantitation demonstrated the reduction of γ-crystallin-bound Cu2+ ions to Cu+ under aerobic conditions, while X-ray absorption spectroscopy (XAS) confirmed the presence of linear or trigonal Cu+ binding sites in γ-crystallins. Our EPR and XAS studies revealed that γ-crystallins' Cu2+ reductase activity yields a protein-based free radical that is likely a Tyr-based species in human γD-crystallin. This unique free radical chemistry carried out by distinct redox-active Cu sites in human lens γ-crystallins likely contributes to the mechanism of copper-induced aggregation. In the context of an aging human lens, γ-crystallins could act not only as structural proteins but also as key players for metal and redox homeostasis.

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

In the human eye lenses, the crystallin proteins facilitate transparency, light refraction, as well as UV light protection. A deregulated balanced interplay between α-, ß-, and γ-crystallin can cause cataract. γD-crystallin (hγD) is involved in the energy dissipation of absorbed UV light by energy transfer between aromatic side chains. Early UV-B induced damage of hγD with molecular resolution is studied by solution NMR and fluorescence spectroscopy. hγD modifications are restricted to Tyr 17 and Tyr 29 in the N-terminal domain, where a local unfolding of the hydrophobic core is observed. None of the tryptophan residues assisting fluorescence energy transfer is modified and hγD is remained soluble over month. Investigating isotope-labeled hγD surrounded by eye lens extracts from cataract patients reveals very week interactions of solvent-exposed side chains in the C-terminal hγD domain and some remaining photoprotective properties of the extracts. Hereditary E107A hγD found in the eye lens core of infants developing cataract shows under the here used conditions a thermodynamic stability comparable to the wild type but an increased sensitivity toward UV-B irradiation.

Mercury ions impact the kinetic and thermal stabilities of human lens γ-crystallins via direct metal-protein interactions.

Loss of metal homeostasis may be involved in several age-related diseases, such as cataracts. Cataracts are caused by the aggregation of lens proteins into light-scattering high molecular weight complexes that impair vision. Environmental exposure to heavy metals, such as mercury, is a risk factor for cataract development. Indeed, mercury ions induce the non-amyloid aggregation of human γC- and γS crystallins, while human γD-crystallin is not sensitive to this metal. Using Differential Scanning Calorimetry (DSC), we evaluate the impact of mercury ions on the kinetic stability of the three most abundant human γ-crystallins. The metal/crystallin interactions were characterized using Isothermal Titration Calorimetry (ITC). Human γD-crystallins exhibited kinetic stabilization due to the presence of mercury ions, despite its thermal stability being decreased. In contrast, human γC- and γS-crystallins are both, thermally and kinetically destabilized by this metal, consistent with their sensitivity to mercury-induced aggregation. The interaction of human γ-crystallins with mercury ions is highly exothermic and complex, since the protein interacts with the metal at more than three sites. The isolated domains of human γ-D and its variant with the H22Q mutation were also studied, revealing the importance of these regions in the mercury-induced stabilization by a direct metal-protein interaction.

Network Hamiltonian Models for Unstructured Protein Aggregates, with Application to γD-Crystallin.

Network Hamiltonian models (NHMs) are a framework for topological coarse-graining of protein-protein interactions, in which each node corresponds to a protein, and edges are drawn between nodes representing proteins that are noncovalently bound. Here, this framework is applied to aggregates of γD-crystallin, a structural protein of the eye lens implicated in cataract disease. The NHMs in this study are generated from atomistic simulations of equilibrium distributions of wild-type and the cataract-causing variant W42R in solution, performed by Wong, E. K.; Prytkova, V.; Freites, J. A.; Butts, C. T.; Tobias, D. J. Molecular Mechanism of Aggregation of the Cataract-Related γD-Crystallin W42R Variant from Multiscale Atomistic Simulations. Biochemistry2019, 58 (35), 3691-3699. Network models are shown to successfully reproduce the aggregate size and structure observed in the atomistic simulation, and provide information about the transient protein-protein interactions therein. The system size is scaled from the original 375 monomers to a system of 10000 monomers, revealing a lowering of the upper tail of the aggregate size distribution of the W42R variant. Extrapolation to higher and lower concentrations is also performed. These results provide an example of the utility of NHMs for coarse-grained simulation of protein systems, as well as their ability to scale to large system sizes and high concentrations, reducing computational costs while retaining topological information about the system.

A novel cataract-causing mutation Ile82Met of γA crystallin trends to aggregate with unfolding intermediate.

Cataract is the most common pathogenic ophthalmic disease leading to blindness in children worldwide. Genetic disorder is the leading cause of congenital cataract, among which crystallin mutations have a high incidence. There are few reports on γA-crystallin, one critical member of crystallin superfamilies. In this study, we identified a novel pathogenic mutation (Ile82Met) in γA-crystallin from a three-generation Chinese family with cataract, and investigated the potential molecular mechanism in detail. To elucidate the pathogenic mechanism of I82M mutant, spectroscopic and solubility experiments were performed to determine the difference between the purified γA-crystallin wild type (WT) and I82M mutant under both physiological conditions and environmental stresses (UV irradiation, thermal denaturation or chemical denaturation). The I82M mutant did not affect the secondary/tertiary structure of monomeric γA-crystallin under physiological status, but decreased protein stability and increased aggregatory potency under the stressful treatment. Surprisingly, the chemical denaturation caused I82M to switch from the two-state unfolding of γA-crystallin to three-state unfolding involving an unfolding intermediate. This study expands the genetic variation map of cataract, and provides novel insights into the pathomechanism, in particular, filling in a gap in the understanding of γA-crystallin mutants causing cataract.

The mechanism for thermal-enhanced chaperone-like activity of α-crystallin against UV irradiation-induced aggregation of γD-crystallin.

Exposure to solar UV irradiation damages γ-crystallin, leading to cataract formation via aggregation. α-Crystallin, as a small heat shock protein, efficiently suppresses this irreversible aggregation by selectively binding the denatured γ-crystallin monomer. In this study, liquid chromatography tandem mass spectrometry was used to evaluate UV-325 nm irradiation-induced photodamage of human γD-crystallin in the presence of bovine α-crystallin, atomic force microscope (AFM) and dynamic light scattering (DLS) techniques were used to detect the quaternary structure changes of the α-crystallin oligomer, and Fourier transform infrared spectroscopy and temperature-jump nanosecond time-resolved IR absorbance difference spectroscopy were used to probe the secondary structure changes of bovine α-crystallin. We find that the thermal-induced subunit dissociation of the α-crystallin oligomer involves the breaking of hydrogen bonds at the dimeric interface, leading to three different spectral components at varied temperature regions as resolved from temperature-dependent IR spectra. Under UV-325 nm irradiation, unfolded γD-crystallin binds to the dissociated α-crystallin subunit to form an αγ-complex, then follows the reassociation of the αγ-complex to the partially dissociated α-crystallin oligomer. This prevents the aggregation of denatured γD-crystallin. The formation of the γD-bound α-crystallin oligomer is further confirmed by AFM and DLS analysis, which reveals an obvious size expansion in the reassociated αγ-oligomers. In addition, UV-325 nm irradiation causes a peptide bond cleavage of γD-crystallin at Ala158 in the presence of α-crystallin. Our results suggest a very effective protection mechanism for subunits dissociated from α-crystallin oligomers against UV irradiation-induced aggregation of γD-crystallin, at the expense of a loss of a short C-terminal peptide in γD-crystallin.

Acquired Disorder and Asymmetry in a Domain-Swapped Model for γ-Crystallin Aggregation.

Misfolding and aggregation of proteins occur in many pathological states. Because of the inherent disorder involved, these processes are difficult to study. We attempted to capture aggregation intermediates of γS-crystallin, a highly stable, internally symmetrical monomeric protein, by crystallization under mildly acidic and oxidizing conditions. Here we describe novel oligomerization through strained domain-swapping and partial intermolecular disulfide formation. This forms an octamer built from asymmetric tetramers, each of which comprises an asymmetric pair of twisted, domain-swapped dimers. Each tetramer shows patterns of acquired disorder among subunits, ranging from local loss of secondary structure to regions of intrinsic disorder. The octamer ring is tied together by partial intermolecular disulfide bonds, which may contribute to strain and disorder in the octamer. Oligomerization in this structure is self-limited by the distorted octamer ring. In a more heterogeneous environment, the disordered regions could serve as seeds for cascading interactions with other proteins. Indeed, solubilized protein from crystals retain many features observed in the crystal and are prone to further oligomerization and precipitation. This structure illustrates modes of loss of organized structure and aggregation that are relevant for cataract and for other disorders involving deposition of formerly well-folded proteins.

Deamidation of the human eye lens protein γS-crystallin accelerates oxidative aging.

Cataract, a clouding of the eye lens from protein precipitation, affects millions of people every year. The lens proteins, the crystallins, show extensive post-translational modifications (PTMs) in cataractous lenses. The most common PTMs, deamidation and oxidation, promote crystallin aggregation; however, it is not clear precisely how these PTMs contribute to crystallin insolubilization. Here, we report six crystal structures of the lens protein γS-crystallin (γS): one of the wild-type and five of deamidated γS variants, from three to nine deamidation sites, after sample aging. The deamidation mutations do not change the overall fold of γS; however, increasing deamidation leads to accelerated disulfide-bond formation. Addition of deamidated sites progressively destabilized protein structure, and the deamidated variants display an increased propensity for aggregation. These results suggest that the deamidated variants are useful as models for accelerated aging; the structural changes observed provide support for redox activity of γS-crystallin in the lens.

Insights to Human γD-Crystallin Unfolding by NMR Spectroscopy and Molecular Dynamics Simulations.

Human γD-crystallin (HGDC) is an abundant lens protein residing in the nucleus of the human lens. Aggregation of this and other structural proteins within the lens leads to the development of cataract. Much has been explored on the stability and aggregation of HGDC and where detailed investigation at the atomic resolution was needed, the X-ray structure was used as an initial starting conformer for molecular modeling. In this study, we implemented NMR-solution HGDC structures as starting conformers for molecular dynamics simulations to provide the missing pieces of the puzzle on the very early stages of HGDC unfolding leading up to the domain swap theories proposed by past studies. The high-resolution details of the conformational dynamics also revealed additional insights to possible early intervention for cataractogenesis.

The contribution of individual residues of an aggregative hexapeptide derived from the human γD-crystallin to its amyloidogenicity.

Human γD-crystallin protein is abundant in the lens and is essential for preserving lens transparency. With age the protein may lose its native structure resulting in the formation of cataract. We recently reported an aggregative peptide, 41Gly-Cys-Trp-Met-Leu-Tyr46 from the human γD-crystallin, termed GDC6, exhibiting amyloidogenic properties in vitro. Here, we aimed to determine the contribution of each residue of the GDC6 to its amyloidogenicity. Molecular dynamic (MD) simulations revealed that the residues Trp, Leu, and Tyr played an important role in the amyloidogenicity of GDC6 by facilitating inter-peptide main-chain hydrogen bonds, and π-π interactions. MD predictions were further validated using single-, double- and triple-alanine-substituted GDC6 peptides in which their amyloidogenic propensity was individually evaluated using complementary biophysical techniques including Thioflavin T assay, turbidity assay, CD spectroscopy, and TEM imaging. Results revealed that the substitution of Trp, Leu, and Tyr together by Ala completely abolished aggregation of GDC6 in vitro, highlighting their importance in the amyloidogenicity of GDC6.

Human γS-Crystallin Resists Unfolding Despite Extensive Chemical Modification from Exposure to Ionizing Radiation.

Ionizing radiation has dramatic effects on living organisms, causing damage to proteins, DNA, and other cellular components. γ radiation produces reactive oxygen species (ROS) that damage biological macromolecules. Protein modification due to interactions with hydroxyl radical is one of the most common deleterious effects of radiation. The human eye lens is particularly vulnerable to the effects of ionizing radiation, as it is metabolically inactive and its proteins are not recycled after early development. Therefore, radiation damage accumulates and eventually can lead to cataract formation. Here we explore the impact of γ radiation on a long-lived structural protein. We exposed the human eye lens protein γS-crystallin (HγS) to high doses of γ radiation and investigated the chemical and structural effects. HγS accumulated many post-translational modifications (PTMs), appearing to gain significant oxidative damage. Biochemical assays suggested that cysteines were affected, with the concentration of free thiol reduced with increasing γ radiation exposure. SDS-PAGE analysis showed that irradiated samples form protein-protein cross-links, including nondisulfide covalent bonds. Tandem mass spectrometry on proteolytic digests of irradiated samples revealed that lysine, methionine, tryptophan, leucine, and cysteine were oxidized. Despite these chemical modifications, HγS remained folded past 10.8 kGy of γ irradiation as evidenced by circular dichroism and intrinsic tryptophan fluorescence spectroscopy.