Aquaporin-0-protein interactions elucidated by crosslinking mass spectrometry.

Aquaporin-0 (AQP0) constitutes 50 % of the lens membrane proteome and plays important roles in lens fiber cell adhesion, water permeability, and lens transparency. Previous work has shown that specific proteins, such as calmodulin (CaM), interact with AQP0 to modulate its water permeability; however, these studies often used AQP0 peptides, rather than full-length protein, to probe these interactions. Furthermore, the specific regions of interaction of several known AQP0 interacting partners, i.e. αA and αB-crystallins, and phakinin (CP49) remain unknown. The purpose of this study was to use crosslinking mass spectrometry (XL-MS) to identify interacting proteins with full-length AQP0 in crude lens cortical membrane fractions and to determine the specific protein regions of interaction. Our results demonstrate, for the first time, that the AQP0 N-terminus can engage in protein interactions. Specific regions of interaction are elucidated for several AQP0 interacting partners including phakinin, α-crystallin, connexin-46, and connexin-50. In addition, two new interacting partners, vimentin and connexin-46, were identified.

Methylglyoxal Induced Modifications to Stabilize Therapeutic Proteins: A Review.

Therapeutic proteins are potent, fast-acting drugs that are highly effective in treating various conditions. Medicinal protein usage has increased in the past 10 years, and it will evolve further as we better understand disease molecular pathways. However, it is associated with high processing costs, limited stability, difficulty in being administered as an oral medication, and the inability of large proteins to penetrate tissue and reach their target locations. Many methods have been developed to overcome the problems with the stability and chaperone activity of therapeutic proteins, viz., the addition of external agents (changing the properties of the surrounding solvent by using stabilizing excipients, e.g., amino acids, sugars, polyols) and internal agents (chemical modifications that influence its structural properties, e.g., mutations, glycosylation). However, these methods must completely clear protein instability and chaperone issues. There is still much work to be done on finetuning chaperone proteins to increase their biological efficacy and stability. Methylglyoxal (MGO), a potent dicarbonyl compound, reacts with proteins and forms covalent cross-links. Much research on MGO scavengers has been conducted since they are known to alter protein structure, which may result in alterations in biological activity and stability. MGO is naturally produced within our body, however, its impact on chaperones and protein stability needs to be better understood and seems to vary based on concentration. This review highlights the efforts of several research groups on the effect of MGO on various proteins. It also addresses the impact of MGO on a client protein, α-crystallin, to understand the potential solutions to the protein's chaperone and stability problems.

Butein Inhibits the Glycation of α-Crystallin: An Approach in Prevention of Retinopathy.

The aggregation of lens proteins induced by glycation is one of the key drivers of diabetic retinopathy and development of diabetic cataracts. Moreover, glycation also causes numerous alterations not only to the tertiary structure of lens proteins but also to serum proteins. There are also evidences of covalent crosslinking among lens crystallins resulting in development of cataract. In this article, the inhibitory potential of butein was tested against the glucose induced glycation and the aggregation α-crystallin (α-cry). The results showed that there was inhibition of advanced glycation products (78.28%) and early glycation products (86.30%) following the treatment of butein. Additionally, the presence of butein caused a significant improvement in the tested biochemical markers of glycation. The treatment with butein reduced the free lysine modification to 23.67%. The secondary and tertiary structural distortions of α-cry were also protected. The mechanism of inhibition further investigated at the molecular level using biophysical and computational techniques. The interaction data showed the butein exhibited strong affinity towards the α-cry. The binding event was entropically driven and energetically favourable. The Gibb's free energy of the interaction was found to be -5.99 to -7.17 kcal mol-1. The binding site of butein in α-cry was deciphered by molecular docking and the dynamics was studied using molecular dynamics (MD) simulations. The simulation data showed that butein formed stable complex with α-cry under physiological conditions. Most of the tested parameters from molecular simulations, such as secondary structure, was found to be stable. The data clearly show the potential of butein in inhibiting the glycation induced aggregation of α-cry and hence can be developed as useful inhibitor in the management of diabetic cataract and retinopathy.

Protein Aggregation and Cataract: Role of Age-Related Modifications and Mutations in α-Crystallins.

* The article is published as a part of the Special Issue "Protein Misfolding and Aggregation in Cataract Disorders" (Vol. 87, No. 2). ** To whom correspondence should be addressed. Cataract is a major cause of blindness. Due to the lack of protein turnover, lens proteins accumulate age-related and environmental modifications that alter their native conformation, leading to the formation of aggregation-prone intermediates, as well as insoluble and light-scattering aggregates, thus compromising lens transparency. The lens protein, α-crystallin, is a molecular chaperone that prevents protein aggregation, thereby maintaining lens transparency. However, mutations or post-translational modifications, such as oxidation, deamidation, truncation and crosslinking, can render α-crystallins ineffective and lead to the disease exacerbation. Here, we describe such mutations and alterations, as well as their consequences. Age-related modifications in α-crystallins affect their structure, oligomerization, and chaperone function. Mutations in α-crystallins can lead to the aggregation/intracellular inclusions attributable to the perturbation of structure and oligomeric assembly and resulting in the rearrangement of aggregation-prone regions. Such rearrangements can lead to the exposure of hitherto buried aggregation-prone regions, thereby populating aggregation-prone state(s) and facilitating amorphous/amyloid aggregation and/or inappropriate interactions with cellular components. Investigations of the mutation-induced changes in the structure, oligomer assembly, aggregation mechanisms, and interactomes of α-crystallins will be useful in fighting protein aggregation-related diseases.

Biochemistry of Eye Lens in the Norm and in Cataractogenesis.

The absence of cellular organelles in fiber cells and very high cytoplasmic protein concentration (up to 900 mg/ml) minimize light scattering in the lens and ensure its transparency. Low oxygen concentration, powerful defense systems (antioxidants, antioxidant enzymes, chaperone-like protein alpha-crystallin, etc.) maintain lens transparency. On the other hand, the ability of crystallins to accumulate age-associated post-translational modifications, which reduce the resistance of lens proteins to oxidative stress, is an important factor contributing to the cataract formation. Here, we suggest a mechanism of cataractogenesis common for the action of different cataractogenic factors, such as age, radiation, ultraviolet light, diabetes, etc. Exposure to these factors leads to the damage and death of lens epithelium, which allows oxygen to penetrate into the lens through the gaps in the epithelial layer and cause oxidative damage to crystallins, resulting in protein denaturation, aggregation, and formation of multilamellar bodies (the main cause of lens opacification). The review discusses various approaches to the inhibition of lens opacification (cataract development), in particular, a combined use of antioxidants and compounds enhancing the chaperone-like properties of alpha-crystallin. We also discuss the paradox of high efficiency of anti-cataract drugs in laboratory settings with the lack of their clinical effect, which might be due to the late use of the drugs at the stage, when the opacification has already formed. A probable solution to this situation will be development of new diagnostic methods that will allow to predict the emergence of cataract long before the manifestation of its clinical signs and to start early preventive treatment.

Effect of Sorbitol on Alpha-Crystallin Structure and Function.

Loss of eye lens transparency due to cataract is the leading cause of blindness all over the world. While aggregation of lens crystallins is the most common endpoint in various types of cataracts, chaperone-like activity (CLA) of α-crystallin preventing protein aggregation is considered to be important for maintaining the eye lens transparency. Osmotic stress due to increased accumulation of sorbitol under hyperglycemic conditions is believed to be one of the mechanisms for diabetic cataract. In addition, compromised CLA of α-crystallin in diabetic cataract has been reported. However, the effect of sorbitol on the structure and function of α-crystallin has not been elucidated yet. Hence, in the present exploratory study, we described the effect of varying concentrations of sorbitol on the structure and function of α-crystallin. Alpha-crystallin purified from the rat lens was incubated with varying concentrations of sorbitol in the dark under sterile conditions for up to 5 days. At the end of incubation, structural properties and CLA were evaluated by spectroscopic methods. Interestingly, different concentrations of sorbitol showed contrasting results: at lower concentrations (5 and 50 mM) there was a decrease in CLA and subtle alterations in secondary and tertiary structure but not at higher concentrations (500 mM). Though, these results shed a light on the effect of sorbitol on α-crystallin structure-function, further studies are required to understand the mechanism of the observed effects and their implication to cataractogenesis.

Ameliorative effect of pioglitazone on glucose induced glycation of α-crystallin: Management of complications associated with diabetic retinopathy.

The glycation and aggregation of lens proteins significantly contribute to the onset of diabetic cataracts as well as the retinopathy. The glycation exerts numerous alterations in the tertiary structural of proteins. Moreover, the covalent crosslinking of lens crystallins also contribute to the cataract formation. In this article, the effect of pioglitazone on glucose induced glycation and aggregation α-crystallin was examined. A remarkable inhibition of early glycation products (~80%) and advanced glycation products (~75%) was recorded by the treatment of pioglitazone. There was >75% recovery in biochemical marker (carbonyl content). The presence of 150 µM of pioglitazone reduced the free lysine modifications to 35%. Treatment of pioglitazone also protected the secondary structural alterations induced by glycation and inhibited the formation of protein aggregates. The interaction studies showed that pioglitazone interacted with α-crystallin via moderate binding affinity. The interaction between pioglitazone interacted and α-crystallin was energetically and entropically favourable. The complex of pioglitazone with studied protein stable in which RMSF, Rg, SASA, RMSD, and the secondary structural components was not affected. The findings show antiglycation activity of pioglitazone along with its mechanism of action highlighting the ability of drug to be possibly developed novel as glycation inhibitor.

α-Crystallins in the Vertebrate Eye Lens: Complex Oligomers and Molecular Chaperones.

α-Crystallins are small heat-shock proteins that act as holdase chaperones. In humans, αA-crystallin is expressed only in the eye lens, while αB-crystallin is found in many tissues. α-Crystallins have a central domain flanked by flexible extensions and form dynamic, heterogeneous oligomers. Structural models show that both the C- and N-terminal extensions are important for controlling oligomerization through domain swapping. α-Crystallin prevents aggregation of damaged ß- and γ-crystallins by binding to the client protein using a variety of binding modes. α-Crystallin chaperone activity can be compromised by mutation or posttranslational modifications, leading to protein aggregation and cataract. Because of their high solubility and their ability to form large, functional oligomers, α-crystallins are particularly amenable to structure determination by solid-state nuclear magnetic resonance (NMR) and solution NMR, as well as cryo-electron microscopy.

Protection of ζ-crystallin by α-crystallin under thermal stress.

Cataract is one of the major causes of blindness worldwide. Several factors including post-translational modification, thermal and solar radiations promote cataractogenesis. The camel lens proteins survive very harsh desert conditions and resist cataractogenesis. The folding and aggregation mechanism of camel lens proteins are poorly characterized. The camel lens contains three ubiquitous crystallins (α-, ß-, and γ-crystallin) and a novel protein (ζ-crystallin) in large amounts. In this study, a sequence similarity search of camel α-crystallin with that of other organisms showed that the camel αB-crystallin consists of an extended N-terminal domain. Our results indicate that camel α-crystallin efficiently prevented aggregation of ζ-crystallin, with or without an obligate cofactor up to 89 °C. It performed a quick and efficient holdase function irrespective of the unfolding stage or aggregation. Camel α-crystallin exhibits approximately 20% chaperone activity between 30 and 40 °C and is completely activated above 40 °C. Camel α-crystallin underwent a single reversible thermal transition without loss of ß-sheet secondary structure. Intrinsic tryptophan fluorescence and ANS binding experiments revealed two transitions which corresponded to activation of its chaperone function. In contrast to earlier studies, camel α-crystallin completely protected lens proteins during thermal stress.

l-3,4-Dihydroxyphenylalanine templated anisotropic gold nano/micro-roses as potential disrupters/inhibitors of α-crystallin protein and its gleaned model peptide aggregates.

Cataract, the major cause of blindness worldwide occurs due to the misfolding and aggregation of the protein crystallin, which constitute a major portion of the lens protein. Other than the whole protein crystallin, the peptide sequences generated from crystallin as a result of covalent protein damage have also been shown to possess and foster protein aggregation, which can be established as crystallin aggregation models. Thus, the disaggregation or inhibition of these protein aggregates could be a viable approach to combat cataract and preserve lens proteostasis. Herein, we tried to explore the disruption as well as inhibition of the intact α-crystallin protein and α-crystallin derived model peptide aggregates by l-3,4-dihydroxyphenylalanine (levodopa) coated gold (Au) nano/micro-roses as modulators. Thioflavin T fluorescence enhancement assay, and electron microscopic analysis were being employed to probe the anti-aggregation behavior of the Au nano/micro-roses towards the aggregating α-crystallin peptides/protein. Further, computational studies were performed to reveal the nature of molecular interactions between the levodopa molecule and the α-crystallin derived model peptides. Interestingly, both levodopa coated Au nano/micro-roses were found to be capable of inhibiting as well as preventing the aggregation of the intact α-crystallin protein and other model peptides derived from it.

Asp isomerization increases aggregation of α-crystallin and decreases its chaperone activity in human lens of various ages.

α-Crystallin, comprising 40-50 subunits of αA- and αB-subunits, is a long-lived major soluble chaperone protein in lens. During aging, α-crystallin forms aggregates of high molecular weight (HMW) protein and eventually becomes water-insoluble (WI). Isomerization of Asp in α-crystallin has been proposed as a trigger of protein aggregation, ultimately leading to cataract formation. Here, we have investigated the relationship between protein aggregation and Asp isomerization of αA-crystallin by a series of analyses of the soluble α-crystallin, HMW and WI fractions from human lens samples of different ages (10-76 years). Analytical ultracentrifugation showed that the HMW fraction had a peak sedimentation coefficient of 40 S and a wide distribution of values (10-450 S) for lens of all ages, whereas the α-crystallin had a much smaller peak sedimentation coefficient (10-20 S) and was less heterogeneous, regardless of lens age. Measurement of the ratio of isomers (Lα-, Lß-, Dα-, Dß-) at Asp58, Asp91/92 and Asp151 in αA-crystallin by liquid chromatography-mass spectrometry showed that the proportion of isomers at all three sites increased in order of aggregation level (α-crystallin < HMW < WI fractions). Among the abnormal isomers of Asp58 and Asp151, Dß-isomers were predominant with a very few exceptions. Notably, the chaperone activity of HMW protein was minimal for lens of all ages, whereas that of α-crystallin decreased with increasing lens age. Thus, abnormal aggregation caused by Asp isomerization might contribute to the loss of chaperone activity of α-crystallin in aged human lens.

Carrier-free self-built aspirin nanorods as anti-aggregation agents towards alpha-crystallin-derived peptide aggregates: potential implications in non-invasive cataract therapy.

The aggregation of the α-crystallin protein is the pathological hallmark of cataract. In the current work, peptide fragments derived from native α-crystallin were synthesized and explored as a peptide-based crystallin aggregation model towards cataract. The anti-aggregation potential of aspirin was evaluated towards these peptide-generated aggregates as well as towards the α-crystallin aggregate. The results demonstrated that aspirin had the capacity to inhibit crystallin and crystallin-derived peptide aggregation and could act as a potential therapeutic agent in mitigating cataract. Computational studies were also carried out to study the interaction between the model peptides and aspirin. The results revealed the existence of molecular interactions between the peptides and aspirin, which had a significant impact on the secondary structure of the peptides and potentially modulated their assembly and aggregation behavior. The formation of self-built aspirin nanorods was also explored and their ability to inhibit the aggregation of model cataract peptides and α-crystallin aggregation was validated. These findings open up the possibility of using small molecule-based nanotherapeutics for cataract merely through topical applications, which can be beneficial to cataract patients.

Isomerization as the secret Achilles' heel of long-lived proteins.

Crystallin proteins, the dominant constituents of the eye lens, are prototypes of long-lived proteins. Such proteins can accumulate harmful modifications over their life span that render them prone to aggregation, which, in the case of lens crystallin, contributes to cataract formation. Lyon et al. now explore the structural and functional consequences of amino acid isomerization in α-crystallins using mass spectrometry, molecular dynamics simulations, and other strategies. Their results highlight the potential deleterious effects of these under-detected modifications on protein structural integrity and function.

Prevention of α-crystallin glycation and aggregation using l-lysine results in the inhibition of in vitro catalase heat-induced-aggregation and suppression of cataract formation in the diabetic rat.

The principle role of α-crystallin is chaperoning activity that protect s other proteins against different stresses. High glucose concentration induces the osmotic stress and results in biomacromolecules glycation, which is subsequently cause their conformational and functional changes. Here, the roles of l-lysine (Lys) on the prevention of α-crystallin glycation in both in vitro and in vivo conditions are investigated. The catalase (CAT) activity was considered as a marker of α-crystallin functionality in both conditions. Streptozotocin-induced diabetic rats were treated with 0.1% of the Lys in drinking water. The purified α-crystallin was also incubated with glucose, in the presence or absence of the Lys and its structure-function was compared. The results showed that the visual cataract score was significantly lower in the diabetic rats treated with Lys. After Lys treatment, CAT, superoxide dismutase, aldose reductase and other biochemical parameters in the lens and serum of the diabetic rats returned to the normal value. Formation of the advanced glycation endproducts (AGEs), protein cross-linking, and the hydrophobicity of α-crystallin were changed due to glycation, but they were reversed by Lys treatment. The glycated α-crystallin lost its chaperone activity against heat denatured-CAT, but in the presence of Lys, it preserved its activity and prevented CAT aggregation. In conclusion, Lys treatment significantly inhibited the progression of diabetic cataract in rats. These effects were due to the Lys antiglycating and antioxidant effects, in addition to its protective effect against α-crystallin chaperoning activity.

O-GlcNAc Site Mapping by Using a Combination of Chemoenzymatic Labeling, Copper-Free Click Chemistry, Reductive Cleavage, and Electron-Transfer Dissociation Mass Spectrometry.

As a dynamic post-translational modification, O-linked ß- N-acetylglucosamine ( O-GlcNAc) modification (i.e., O-GlcNAcylation) of proteins regulates many biological processes involving cellular metabolism and signaling. However, O-GlcNAc site mapping, a prerequisite for site-specific functional characterization, has been a challenge since its discovery. Herein we present a novel method for O-GlcNAc enrichment and site mapping. In this method, the O-GlcNAc moiety on peptides was labeled with UDP-GalNAz followed by copper-free azide-alkyne cycloaddition with a multifunctional reagent bearing a terminal cyclooctyne, a disulfide bridge, and a biotin handle. The tagged peptides were then released from NeutrAvidin beads upon reductant treatment, alkylated with (3-acrylamidopropyl)trimethylammonium chloride, and subjected to electron-transfer dissociation mass spectrometry analysis. After validation by using standard synthetic peptide gCTD and model protein α-crystallin, such an approach was applied to the site mapping of overexpressed TGF-ß-activated kinase 1/MAP3K7 binding protein 2 (TAB2), with four O-GlcNAc sites unambiguously identified. Our method provides a promising tool for the site-specific characterization of O-GlcNAcylation of important proteins.

Genome-wide identification of evolutionarily conserved Small Heat-Shock and eight other proteins bearing α-crystallin domain-like in kinetoplastid protists.

Small Heat-Shock Proteins (sHSPs) and other proteins bearing alpha-crystallin domains (ACD) participate in defense against heat and oxidative stress and play important roles in cell cycle, cytoskeleton dynamics, and immunological and pathological mechanisms in eukaryotes. However, little is known about these proteins in early-diverging lineages of protists such as the kinetoplastids. Here, ACD-like proteins (ACDp) were investigated in genomes of 61 species of 12 kinetoplastid genera, including Trypanosoma spp. (23 species of mammals, reptiles and frogs), Leishmania spp. (mammals and lizards), trypanosomatids of insects, Phytomonas spp. of plants, and bodonids. Comparison of ACDps based on domain architecture, predicted tertiary structure, phylogeny and genome organization reveals a kinetoplastid evolutionarily conserved repertoire, which diversified prior to trypanosomatid adaptation to parasitic life. We identified 9 ACDp orthologs classified in 8 families of TryACD: four previously recognized (HSP20, Tryp23A, Tryp23B and ATOM69), and four characterized for the first time in kinetoplastids (TryACDP, TrySGT1, TryDYX1C1 and TryNudC). A single copy of each ortholog was identified in each genome alongside TryNudC1/TrypNudC2 homologs and, overall, ACDPs were under strong selection pressures at main phylogenetic lineages. Transcripts of all ACDPs were identified across the life stages of T. cruzi, T. brucei and Leishmania spp., but proteomic profiles suggested that most ACDPs may be species- and stage-regulated. Our findings establish the basis for functional studies, and provided evolutionary and structural support for an underestimated repertoire of ACDps in the kinetoplastids.

Localization of αA-Crystallin in Rat Retinal Müller Glial Cells and Photoreceptors.

Transparent cells in the vertebrate optical tract, such as lens fiber cells and corneal epithelium cells, have specialized proteins that somehow permit only a low level of light scattering in their cytoplasm. It has been shown that both cell types contain (1) beaded intermediate filaments as well as (2) α-crystallin globulins. It is known that genetic and chemical alterations to these specialized proteins induce cytoplasmic opaqueness and visual complications. Crystallins were described previously in the retinal Müller cells of frogs. In the present work, using immunocytochemistry, fluorescence confocal imaging, and immuno-electron microscopy, we found that αA-crystallins are present in the cytoplasm of retinal Müller cells and in the photoreceptors of rats. Given that Müller glial cells were recently described as "living light guides" as were photoreceptors previously, we suggest that αA-crystallins, as in other highly transparent cells, allow Müller cells and photoreceptors to minimize intraretinal scattering during retinal light transmission.

[On-line analysis of T and B cell epitopes of α-crystallin (Rv2031c) derived from Mycobacterium tuberculosis].

Objective To predict the epitopes of Mycobacterium tuberculosis dormancy-related protein α-crystallin (Rv2031c). Methods The homology between Rv2031c and human proteins was analyzed online by BLAST alignment. B- and T-cell epitopes of Rv2031c were predicted by Protean of DNAStar software. RANKPEP and SYPEPITHI were used to predict the epitopes of T helper (Th) cells; while SYFPEPI, BIMAS, and NetCTL were used to predict the epitopes of cytotoxic T lymphocytes (CTLs). Results Rv2031c had low homology with human proteins. Eight potential epitopes of B-cells, 7 epitopes of Th cells and 3 epitopes of CTLs were predicted in Rv2031c. Conclusion Rv2031c, which has many potential epitopes of Th cells, CTLs and B-cells, is expected to be a promising candidate for the development of tuberculosis vaccines.

3D structure of the native α-crystallin from bovine eye lens.

α-Crystallin is the major eye lens protein that has been shown to support lens transparency by preventing the aggregation of lens proteins. The 3D structure of α-crystallin is largely unknown. Electron microscopy, single-particle 3D reconstruction, size exclusion chromatography, dynamic light scattering, and analytical ultracentrifugation were used to study the structure of the native α-crystallin. Native α-crystallin has a wide distribution in size. The shape of mass distribution is temperature-dependent, but the oligomers with a sedimentation coefficient of ~22 S (750-830 kDa) strongly prevailed at all temperatures used. A 3D model of native α-crystallin with resolution of ~2 nm was created. The model is asymmetrical, has an elongated bean-like shape 13 × 19 nm with a dense core and filamentous "kernel". It does not contain a central cavity. The majority of α-crystallin particles regardless of experimental conditions are 13 × 19 nm, which corresponds to 22S sedimentation coefficient, hydrodynamic diameter 20 nm and mass of 750-830 kD. These particles are in dynamic equilibrium with particles of smaller and larger sizes.

Differences in α-Crystallin isomerization reveal the activity of protein isoaspartyl methyltransferase (PIMT) in the nucleus and cortex of human lenses.

Although it is well-known that protein turnover essentially stops in mature lens fiber cells, mapping out the ensuing protein degradation and its effects on lens function over time remains challenging. In particular, isomerization is a common, spontaneous post-translational modification that occurs over long timescales and generates products invisible to most analytical methods. Nevertheless, isomerization can significantly impact protein structure, function, and solubility, which are all necessary to maintain clarity and proper refractive index within the lens. Herein, we examine the degree of isomerization occurring in crystallin proteins in the human eye lens as a function of both age and location within the lens. A novel mass spectrometric technique leveraging radical chemistry enables detailed characterization of proteins extracted from the cortex and nucleus of the lens. It is observed that the degree of isomerization increases significantly between the cortex and nucleus and between water-soluble and water-insoluble fractions. Interestingly, the abundance of L-isoAsp is low in the water-soluble cortex despite being the dominant product generated by isomerization of Asp in vitro, suggesting that Protein L-isoaspartyl methyltransferase (PIMT) is active in the cortex and suppresses the accumulation of L-isoAsp. The abundance of L-isoAsp increases dramatically in the nucleus, revealing that PIMT activity decreases over time in the center of the lens. In addition, the growth of L-isoAsp in the nuclear fraction suggests protein isomerization continues within the nucleus, despite the fact that most of the protein within the nucleus has become insoluble. Additionally, it is demonstrated that sequential Asp residues lead to isomerization hotspots in human crystallin proteins and that the isomerization profiles for αA and αB crystallin are notably different. Although αA is more prone to isomerization, αB loses solubility more rapidly upon modification. These differences are likely related to the distribution of Asp residues within αA and αB, which are in turn connected to refractive index. The high Asp content of αA is a hazard in terms of isomerization and aging, but it serves to enhance the refractive index of αA relative to αB, and may explain why αA is only found in the eye.