Human γS-crystallin Research Articles

Molecular dynamics of the chaperone peptide mini-αA-crystallin, and its interaction with human γS-crystallin

Molecular dynamics of the chaperone peptide mini-αA-crystallin, and its interaction with human γS-crystallin

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 βγ-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.

Impact of hydrogen peroxide on structure, stability, and aggregational properties of human γS-crystallin

Cataract is the leading cause of blindness worldwide. Oxidative stress is one of the known risk factors for agerelated cataracts. The present study was designed to understand the effect of H2O2-induced oxidative stress on human γS-crystallin and its relationship to lens opacification and cataract. Human γS-crystallin cDNA was cloned into the pET-20b vector, overexpressed in BL21 Star (DE3) cells, and was purified using ion-exchange and gel filtration chromatography. The structure, stability, and aggregational properties of human γS-crystallin under H2O2 stress were studied using fluorescence and circular dichroism spectroscopy methods. H2O2 treatment did not show any significant effect on the γS-crystallin secondary structure but showed an effect on its tertiary structure, resulting in N'-formylkynurenine formation. The H2O2-treated sample showed increased surface hydrophobicity, was less stable, and opened its Greek key motifs earlier with a midpoint of thermal unfolding curve (Tm) of 70.2°C compared with untreated γS-crystallin (Tm=71.4°C). The sample treated with H2O2 aggregated earlier in response to heating at 65°C. H2O2-induced oxidative stress alters the tryptophan microenvironment and the surface hydrophobicity of γS-crystallin, and these changes decrease its thermal stability and increase its tendency to aggregate, consistent with its role as a risk factor in age-related cataract.

Partial assignments of tunicate βγ-crystallin structure.

Tunicate Ciona intestinalis (Ci-βγ-crystallin) is a model to study evolution of vertebrates as it has an evolutionary position between the microbial crystallins and the vertebrate lens proteins. The single-domain βγ-crystallin of the tunicate Ci-βγ-crystallin represents the single-domain ancestor of the vertebrate βγ-crystallin. It has been shown that the tunicate Ci-βγ-crystallin has a high affinity to bind to Ca2+ and that this binding greatly stabilizes the protein both thermally and chemically. However, human γS-crystallin doesn't bind to Ca2+ but it stays stable. NMR spectroscopy is used to study the structure and properties of Ci-βγ-crystallin to better understand the stability and evolutionary progression from ancestral to human lens βγ-crystallins. Residual dipolar couplings (RDCs), derived from 15N-1H in-phase/anti-phase (IPAP) heteronuclear single quantum coherence NMR spectra, provide relative orientations among internuclei vectors. These orientations can be used to build the protein backbone structure. RDCs are obtained by weakly aligning proteins in a magnetic field to create an anisotropic condition. The magnetic dipole-dipole interactions no longer average to zero and give measurable dipolar couplings. RDCs are used along with other experimental NMR data for protein structure determination. Xplor-NIH package will be used for protein structure refinement. Xplor-NIH is a popular software package for biomolecular structure determination. The program takes experimental NMR data such as RDCs and other pre-determined theoretical parameters such as covalent geometry (bond lengths and angles) to find the minimum energy structures.

Damage resistance of gamma crystallins: Weathering heat, UV, and ionizing radiation.

The γ-crystallins are structural and refractive proteins of the vertebrate lens. They belong to the βγ-crystallin superfamily, which is characterized by an exceptionally stable double-Greek key fold. In the context of the lens, these proteins must remain stable and soluble for the lifetime of the organism. Recent work in my group focuses on understanding how γ-crystallins maintain functionality in the face of damage caused by aging and oxidation. I will present results on the impact of heat, UV light, and ionizing radiation on human γS-crystallin and cataract-associated variants. We find that the γ-crystallins are not particularly resistant to damage; on the contrary, they appear to be selected for the ability to remain soluble even when the protein surface is significantly modified and transient unfolding intermediates are populated. These have been investigated using a variety of biophysical techniques, including circular dichroism, mass spectrometry, fluorescence, and NMR spectroscopy. In particular, mass spectrometry measurements on irradiated samples show evidence of both deamidation and oxidative damage, while high-resolution NMR data allow us to locate protein regions and specific residues that are involved in conformational changes, altered dynamics, and protein-protein contacts. We will discuss proposed aggregation mechanisms mediated by different types of protein modification, as well as the relevance of these findings to human cataract disease.

Acute doses of ionizing radiation extensively modifies human γS-crystallin without destruction of overall protein fold

The human lens is densely packed with a class of extremely long-lived proteins called crystallins that maintain the transparency and refractivity of the tissue. Cataract, a leading cause of blindness worldwide, is the result of aggregation of these proteins that increases the opacity of the lens. Ionizing radiation has been linked to early onset of cataract; however, the mechanism of radiation-induced cataract remains unclear. Here, we exposed a human lens crystallin, gammaS (HgammaS), to acute doses of gamma irradiation and evaluated the resulting chemical and structural changes.

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.

Bayesian analysis of static light scattering data for globular proteins.

Static light scattering is a popular physical chemistry technique that enables calculation of physical attributes such as the radius of gyration and the second virial coefficient for a macromolecule (e.g., a polymer or a protein) in solution. The second virial coefficient is a physical quantity that characterizes the magnitude and sign of pairwise interactions between particles, and hence is related to aggregation propensity, a property of considerable scientific and practical interest. Estimating the second virial coefficient from experimental data is challenging due both to the degree of precision required and the complexity of the error structure involved. In contrast to conventional approaches based on heuristic ordinary least squares estimates, Bayesian inference for the second virial coefficient allows explicit modeling of error processes, incorporation of prior information, and the ability to directly test competing physical models. Here, we introduce a fully Bayesian model for static light scattering experiments on small-particle systems, with joint inference for concentration, index of refraction, oligomer size, and the second virial coefficient. We apply our proposed model to study the aggregation behavior of hen egg-white lysozyme and human γS-crystallin using in-house experimental data. Based on these observations, we also perform a simulation study on the primary drivers of uncertainty in this family of experiments, showing in particular the potential for improved monitoring and control of concentration to aid inference.

ATP differentially antagonizes the crowding-induced destabilization of human γS-crystallin and its four cataract-causing mutants

αβγ-crystallins account for ∼90% of ocular proteins in lens with concentrations ≥400 mg/ml, which has to be soluble for the whole life-span and their aggregation results in cataract. So far, four cataract-causing mutants G18V, D26G, S39C and V42 M have been identified for human γS-crystallin. Mysteriously, lens maintains ATP concentrations of 3–7 mM despite being a metabolically-quiescent organ. Here by DSF and NMR, we characterized the binding of ATP to three cataract-causing mutants of human γS-crystallin as well as its effect on the solution conformations and thermal stability. The results together decode several novel findings: 1) ATP shows no detectable binding to WT and mutants, as well as no significant alternation of their conformations even at molar ratio of 1:200.2) Cataract-causing mutants show distinctive patterns of the crowding-induced destabilization. 3) ATP differentially antagonizes their crowding-induced destabilization. Our studies suggest that the crowding-induced destabilization of human γS-crystallin is also critically dependent of the hydration shell which could be differentially altered by four mutations. Most unexpectedly, ATP acts as an effective mediator for the protein hydration shell to antagonize the crowding-induced destabilization.

Cataract-causing G18V eliminates the antagonization by ATP against the crowding-induced destabilization of human γS-crystallin

In lens, ∼90% of ocular proteins are αβγ-crystallins with concentrations ≥400 mg/ml, which need to remain soluble for the whole life-span and their aggregation leads to cataract. The G18V mutation of human γS-crystallin causes hereditary childhood-onset cortical cataract. Mysteriously, despite being a metabolically-quiescent organ, lens maintains ATP concentrations of 3–7 mM. Very recently, we found that ATP has no significant binding to γS-crystallin as well as no alternation of its conformation. Nevertheless, ATP antagonizes the crowding-induced destabilization of γS-crystallin even at 1:1, most likely by interacting with the hydration shell. Here by DSF and NMR, we characterized the effect of ATP on binding, conformation, stability of G18V γS-crystallin and its interactions with α-crystallin. The results reveal: 1) G18V significantly accelerates the crowding-induced destabilization with Tm of 67 °C reduced to 50.5 °C at 1 mM. 2) Most unexpectedly, G18V almost completely eliminates the antagonizing effect of ATP against the crowding-induced destabilization. 3) ATP shows no significant effect on the interactions of α-crystallin with both WT and G18V γS-crystallin. Results together decode for the first time that G18V causes cataract not only by accelerating the crowding-induced destabilization, but also by eliminating the antagonizing effect of ATP against the crowding-induced destabilization.

Human γS-Crystallin-Copper Binding Helps Buffer against Aggregation Caused by Oxidative Damage.

Divalent metal cations can play a role in protein aggregation diseases, including cataract. Here we compare the aggregation of human γS-crystallin, a key structural protein of the eye lens, via mutagenesis, ultraviolet light damage, and the addition of metal ions. All three aggregation pathways result in globular, amorphous-looking structures that do not elongate into fibers. We also investigate the molecular mechanism underlying copper(II)-induced aggregation. This work was motivated by the observation that zinc(II)-induced aggregation of γS-crystallin is driven by intermolecular bridging of solvent-accessible cysteine residues, while in contrast, copper(II)-induced aggregation of this protein is exacerbated by the removal of solvent-accessible cysteines via mutation. Here we find that copper(II)-induced aggregation results from a complex mechanism involving multiple interactions with the protein. The initial protein-metal interactions result in the reduction of Cu(II) to Cu(I) with concomitant oxidation of γS-crystallin. In addition to the intermolecular disulfides that represent a starting point for aggregation, intramolecular disulfides also occur in the cysteine loop, a region of the N-terminal domain that was previously found to mediate the early stages of cataract formation. This previously unobserved ability of γS-crystallin to transfer disulfides intramolecularly suggests that it may serve as an oxidation sink for the lens after glutathione levels have become depleted during aging. γS-Crystallin thus serves as the last line of defense against oxidation in the eye lens, a result that underscores the chemical functionality of this protein, which is generally considered to play a purely structural role.

ATP antagonizes the crowding-induced destabilization of the human eye-lens protein γS-crystallin

In lens, αβγ-crystallins accounting for ∼90% of ocular proteins with concentrations >400 mg/ml need to remain soluble for the whole life-span and their aggregation can lead to cataract. Mysteriously, despite being a metabolically-quiescent organ, lens maintains ATP concentrations of 3–7 mM. Very recently, ATP was proposed to hydrotropically prevent aggregation of crystallins but the mechanism remains unexplored. Here by NMR, DLS and DSF, we characterized the association, thermal stability and conformation of the 178-residue human γS-crystallin at concentrations from 2 to 100 mg/ml in the absence and in the presence of ATP. Results together reveal for the first time that ATP does antagonize the crowding-induced destabilization, although it has no significant binding to γS-crystallin as well as no alteration of its conformation. Therefore, ATP prevents aggregation in lens by a novel mechanism, thus rationalizing the fact that declining concentrations of ATP upon being aged is related to age-related cataractogenesis. To restore the normal concentrations of ATP in lens may represent a promising therapeutic strategy to treat aggregation-causing eye diseases.

Human αB-crystallin discriminates between aggregation-prone and function-preserving variants of a client protein

BackgroundThe eye lens crystallins are highly soluble proteins that are required to last the lifespan of an organism due to low protein turnover in the lens. Crystallin aggregation leads to formation of light-scattering aggregates known as cataract. The G18V mutation of human γS-crystallin (γS-G18V), which is associated with childhood-onset cataract, causes structural changes throughout the N-terminal domain and increases aggregation propensity. The holdase chaperone protein αB-crystallin does not interact with wild-type γS-crystallin, but does bind its G18V variant. The specific molecular determinants of αB-crystallin binding to client proteins is incompletely charcterized. Here, a new variant of γS, γS-G18A, was created to test the limits of αB-crystallin selectivity. MethodsMolecular dynamics simulations were used to investigate the structure and dynamics of γS-G18A. The overall fold of γS-G18A was assessed by circular dichroism (CD) spectroscopy and intrinsic tryptophan fluorescence. Its thermal unfolding temperature and aggregation propensity were characterized by CD and DLS, respectively. Solution-state NMR was used to characterize interactions between αB-crystallin and γS-G18A. ResultsγS-G18A exhibits minimal structural changes, but has compromised thermal stability relative to γS-WT. The placement of alanine, rather than valine, at this highly conserved glycine position produces minor changes in hydrophobic surface exposure. However, human αB-crystallin does not bind the G18A variant, in contrast to previous observations for γS-G18V, which aggregates at physiological temperature. ConclusionsαB-crystallin is capable of distinguishing between aggregation-prone and function-preserving variants, and recognizing the transient unfolding or minor conformers that lead to aggregation in the disease-related variant. General significanceHuman αB-crystallin distinguishes between highly similar variants of a structural crystallin, binding the cataract-related γS-G18V variant, but not the function-preserving γS-G18A variant, which is monomeric at physiological temperature.

A Molecular Dynamics Perspective To Identify Precursors to Aggregation in Human γS-Crystallin Unravels the Mechanism of Childhood Cataracts.

Despite the increasing health risk from infantile cataracts, identifying the mechanism of this disease remains a challenge due to a lack of structural investigations using experimental and computational approaches. Mutations in human γS-crystallin are contingent with childhood cataracts. Our recent high-resolution structural studies using solution NMR spectroscopy established the key role of the G57W mutation in human γS-crystallin (abbreviated hereafter as γS-G57W), promoting structural instability. In order to design therapeutics to delay or upset congenital cataracts, the characterization of the precursors to γS-G57W aggregation is indispensable. In this endeavor, we present microsecond long unbiased atomistic molecular dynamics simulations and principal component analyses that unfold insights into lens crystallin aggregation. An enhanced sampling metadynamics approach was further employed to systematically unravel the molecular dynamics underlying crucial interdomain contacts. Taken together, our experiment-guided computational study in this paper led to the identification of domain-swapped intermediates in γS-G57W to atomic resolution with insights into the aggregation of lens crystallins causing childhood cataracts for the first time with functional consequences.

Divalent Cations and the Divergence of βγ-Crystallin Function.

The βγ-crystallin superfamily contains both β- and γ-crystallins of the vertebrate eye lens and the microbial calcium-binding proteins, all of which are characterized by a common double-Greek key domain structure. The vertebrate βγ-crystallins are long-lived structural proteins that refract light onto the retina. In contrast, the microbial βγ-crystallins bind calcium ions. The βγ-crystallin from the tunicate Ciona intestinalis (Ci-βγ) provides a potential link between these two functions. It binds calcium with high affinity and is found in a light-sensitive sensory organ that is highly enriched in metal ions. Thus, Ci-βγ is valuable for investigating the evolution of the βγ-crystallin fold away from calcium binding and toward stability in the apo form as part of the vertebrate lens. Here, we investigate the effect of Ca2+ and other divalent cations on the stability and aggregation propensity of Ci-βγ and human γS-crystallin (HγS). Beyond Ca2+, Ci-βγ is capable of coordinating Mg2+, Sr2+, Co2+, Mn2+, Ni2+, and Zn2+, although only Sr2+ is bound with comparable affinity to its preferred metal ion. The extent to which the tested divalent cations stabilize Ci-βγ structure correlates strongly with ionic radius. In contrast, none of the tested divalent cations improved the stability of HγS, and some of them induced aggregation. Zn2+, Ni2+, and Co2+ induce aggregation by interacting with cysteine residues, whereas Cu2+-mediated aggregation proceeds via a different binding site.

Altered Protein Dynamics and Increased Aggregation of Human γS-Crystallin Due to Cataract-Associated Deamidations.

Deamidation is a major age-related modification in the human lens that is highly prevalent in crystallins isolated from the insoluble fraction of cataractous lenses and also causes protein aggregation in vitro. However, the mechanism by which deamidation causes proteins to become insoluble is not known because only subtle structural changes were observed in vitro. We have identified Asn14 and Asn76 of γS-crystallin as highly deamidated in insoluble proteins isolated from aged lenses. These sites are on the surface of the N-terminal domain and were mimicked by replacing the Asn with Asp residues in order to generate recombinant human γS and deamidated mutants. Both N14D and N76D had increased light scattering compared to wild-type γS (WT) and increased aggregation during thermal-induced denaturation. Aggregation was enhanced by oxidized glutathione, suggesting deamidation may increase susceptibility to form disulfide bonds. These changes were correlated to changes in protein dynamics determined by NMR spectroscopy. Heteronuclear NMR spectroscopy was used to measure amide hydrogen exchange and 15N relaxation dynamics to identify regions with increased dynamics compared to γS WT. Residue-specific changes in solvent accessibility and dynamics were both near and distant from the sites of deamidation, suggesting that deamidation had both local and global effects on the protein structure at slow (ms to s) and fast (μs to ps) time scales. Thus, a potential mechanism for γS deamidation-induced insolubilization in cataractous lenses is altered dynamics due to local regions of unfolding and increased flexibility in both the N- and C-terminal domains particularly at surface helices. This conformational flexibility increases the likelihood of aggregation, which would be enhanced in the oxidizing cytoplasm of the aged and cataractous lens. The NMR data combined with the in vivo insolubility and in vitro aggregation findings support a model that deamidation drives changes in protein dynamics that facilitate protein aggregation associated with cataracts.

Structural studies on the individual domains of human γS-crystallin and its G57W mutant unfolds mechanistic insights into childhood cataracts

Inter-domain interactions tune the exceptional stability of human γS-crystallin (γS-WT) in the eye lens, which lasts a lifetime with no protein turnover. Our recent NMR studies revealed the key role of G57W mutation in γS-WT, as the familial determinate of childhood cataracts. As the unusually exposed W57 is near the inter-domain interface, a recurring theme of this study is the upsetting of inter-domain contacts exposing hydrophobic patches, which may initiate aggregation at crystallin concentrations not so surprising in the eye lens. In this endeavour, to untangle the mechanistic pathways triggering aggregation in the cataract variant γS-G57W, we undertook high-resolution structural characterization of isolated domains vis-a-vis full length γS-crystallin. Here we report for the first time, thermodynamic and kinetic determinants of structural stability with their eccentric shifts under pathological stress employing sophisticated spectroscopy techniques. We propose that domain interface acts as an intrinsic stabilizer for the otherwise floppy N-terminal domain in γS-G57W than in γS-WT where it serves an extrinsic role. Our results present a residue resolved quantitative analysis for differential domain stabilities from non-linear temperature coefficients of 1HN chemical shifts using solution NMR spectroscopy. Consistent with the Ca2+-binding episode that lasted poorly for human lens crystallins, our results show that disease mutants attenuate it further and completely silence it in extreme cases. Overall, our study provides a compelling evidence for the diverse structural evolution of lens crystallins elucidating molecular details to apprehend lens opacification and suggests the scope of therapeutics in reducing the global trauma of cataracts.

On identifying low energy conformational excited states with differential ruggedness in human γS-crystallin promoting severe infantile cataracts

Transient excited states in proteins can be accurately probed from temperature dependence of amide proton (1HN) chemical shifts displaying significant curvatures. Characterizing these near-native alternative states is of high therapeutic relevance in conformational diseases wherein missense mutations promote structural instability that leads to conformational heterogeneity. Extending the structure-function paradigm from physiology to pathology, we recently reported the solution NMR structure and dynamics of a severe congenital cataract variant, G57W of human γS-crystallin (abbreviated as γS-G57W) which is resistant towards crystallization. In an endeavour to explore the functional consequences of this mutation, here we report for the first time, native state conformational ruggedness in human γS-G57W as compared to its wild-type counterpart from residue resolved nonlinear temperature dependence of backbone 1HN chemical shifts using solution NMR spectroscopy. Our calculations suggest that the simulated chemical shift curvatures are indicative of low energy excited states within 2–4 kcal mol−1 from the native state. Residues accessing alternative conformations populate the N-terminal domain of γS-G57W more than its C-terminal counterpart. Collectively, curvatures retaining native state ensemble on mild denaturation suggest that the free energy landscape in human γS-G57W at the bottom of the folding funnel is sufficiently robust and malleable against such perturbations. Overall, this critical study highlights the functional aspects of such structural malleability promoting infantile cataracts as a global health risk marker.

Enhanced H/D exchange unravels sequential structural excursions in G57W variant of human γS-crystallin with pro-cataractogenic conformations

Our two recent reports on the high resolution NMR structure and conformational dynamics of G57W variant of human γS-crystallin (abbreviated as γS-G57W) causing severe infantile cataracts, revealed slackening of its N-terminal domain with enhanced local conformational dynamics attributed to mutation. Exploring the biochemistry of infantile cataracts in detail, here we studied structural unfolding in both human γS-WT and γS-G57W at residue level resolution using solution NMR spectroscopy and chemical kinetics and characterized the molecular intermediates with functional consequences. We report, for the first time, that human γS-crystallin unfolds sequentially under H/D exchange. This communication forms the first experimental evidence for non-concerted destabilization of structural foldon units in human γS-G57W. Residues contributing to the compact fold and structural stability exchanged their amide protons with deuterons more readily in γS-G57W compared to γS-WT, displaying differential free energies of exchange. Overall, our results establish a direct conformational link between the structure, dynamics, design and function in human γS-crystallin such that the G57W cataract variant promotes enhanced structural excursions concomitant with increased instability, elucidating very crucial molecular details of cataract formation affecting infants across the globe.

Conformational dynamics study on human γS-crystallin as an efficient route to childhood blindness

Single point mutants of human γS-crystallin cause dominant congenital cataracts, a recent one of which involves the substitution of highly conserved glycine at 57th position with a bulkier tryptophan. Our high-resolution 3D structure of this G57W mutant (abbreviated hereafter as γS-G57W), reported recently revealed site-specific structural perturbations with higher aggregation and lower stability compared to its wild-type; a structural feature associated with important functional and therapeutic consequences. In this communication, we report for the first time, residue resolved conformational dynamics in both γS-WT and γS-G57W using solution NMR spectroscopy, and suggest how these differences could crucially affect the biochemistry of the mutant. Guided by our critical structural investigations, extensive conformational dynamics and biophysical studies presented here show that loss of structural stability arises from enhanced dynamics in Greek key motif 2 inducing flexibility in the N-terminal domain as opposed to its structurally unperturbed C-terminal counterpart. NMR spectral density correlations and internal dynamics comparisons with the wild-type suggest that the overall thermodynamic instability propagates from the mutated N-terminal β4-β5 loop providing a residue level understanding of the structural changes associated with this early onset of lens opacification. Our results highlight the vital role of conserved Greek key motifs in conferring structural stability to crystallins and provide crucial molecular insights into crystallin aggregation in the eye lens, which triggers cataract formation in children. Overall, this critical study provides a residue level understanding of how conformational changes affect the structure and function of crystallins in particular and proteins in general, during health and disease.