Deamidation in human lens betaB2-crystallin destabilizes the dimer

Two major determinants of the transparency of the lens are protein-protein interactions and stability of the crystallins, the structural proteins in the lens. betaB2 is the most abundant beta-crystallin in the human lens and is important in formation of the complex interactions of lens crystallins. betaB2 readily forms a homodimer in vitro, with interacting residues across the monomer-monomer interface conserved among beta-crystallins. Due to their long life spans, crystallins undergo an unusually large number of modifications, with deamidation being a major factor. In this study the effects of two potential deamidation sites at the monomer-monomer interface on dimer formation and stability were determined. Glutamic acid substitutions were constructed to mimic the effects of previously reported deamidations at Q162 in the C-terminal domain and at Q70, its N-terminal homologue. The mutants had a nativelike secondary structure similar to that of wild type betaB2 with differences in tertiary structure for the double mutant, Q70E/Q162E. Multiangle light scattering and quasi-elastic light scattering experiments showed that dimer formation was not interrupted. In contrast, equilibrium unfolding and refolding in urea showed destabilization of the mutants, with an inflection in the transition of unfolding for the double mutant suggesting a distinct intermediate. These results suggest that deamidation at critical sites destabilizes betaB2 and may disrupt the function of betaB2 in the lens.     

Deamidation alters the structure and decreases the stability of human lens betaA3-crystallin

According to the World Health Organization, cataracts account for half of the blindness in the world, with the majority occurring in developing countries. A cataract is a clouding of the lens of the eye due to light scattering of precipitated lens proteins or aberrant cellular debris. The major proteins in the lens are crystallins, and they are extensively deamidated during aging and cataracts. Deamidation has been detected at the domain and monomer interfaces of several crystallins during aging. The purpose of this study was to determine the effects of two potential deamidation sites at the predicted interface of the betaA3-crystallin dimer on its structure and stability. The glutamine residues at the reported in vivo deamidation sites of Q180 in the C-terminal domain and at the homologous site Q85 in the N-terminal domain were substituted with glutamic acid residues by site-directed mutagenesis. Far-UV and near-UV circular dichroism spectroscopy indicated that there were subtle differences in the secondary structure and more notable differences in the tertiary structure of the mutant proteins compared to that of the wild type betaA3-crystallin. The Q85E/Q180E mutant also was more susceptible to enzymatic digestion, suggesting increased solvent accessibility. These structural changes in the deamidated mutants led to decreased stability during unfolding in urea and increased precipitation during heat denaturation. When simulating deamidation at both residues, there was a further decrease in stability and loss of cooperativity. However, multiangle-light scattering and quasi-elastic light scattering experiments showed that dimer formation was not disrupted, nor did higher-order oligomers form. These results suggest that introducing charges at the predicted domain interface in the betaA3 homodimer may contribute to the insolubilization of lens crystallins or favor other, more stable, crystallin subunit interactions.     

Cumulative deamidations of the major lens protein γS‐crystallin increase its aggregation during unfolding and oxidation

Age‐related lens cataract is the major cause of blindness worldwide. The mechanisms whereby crystallins, the predominant lens proteins, assemble into large aggregates that scatter light within the lens, and cause cataract, are poorly understood. Due to the lack of protein turnover in the lens, crystallins are long‐lived. A major crystallin, γS, is heavily modified by deamidation, in particular at surface‐exposed N14, N76, and N143 to introduce negative charges. In this present study, deamidated γS was mimicked by mutation with aspartate at these sites and the effect on biophysical properties of γS was assessed via dynamic light scattering, chemical and thermal denaturation, hydrogen‐deuterium exchange, and susceptibility to disulfide cross‐linking. Compared with wild type γS, a small population of each deamidated mutant aggregated rapidly into large, light‐scattering species that contributed significantly to the total scattering. Under partially denaturing conditions in guanidine hydrochloride or elevated temperature, deamidation led to more rapid unfolding and aggregation and increased susceptibility to oxidation. The triple mutant was further destabilized, suggesting that the effects of deamidation were cumulative. Molecular dynamics simulations predicted that deamidation augments the conformational dynamics of γS. We suggest that these perturbations disrupt the native disulfide arrangement of γS and promote the formation of disulfide‐linked aggregates. The lens‐specific chaperone αA‐crystallin was poor at preventing the aggregation of the triple mutant. It is concluded that surface deamidations cause minimal structural disruption individually, but cumulatively they progressively destabilize γS‐crystallin leading to unfolding and aggregation, as occurs in aged and cataractous lenses.     

Deamidation destabilizes and triggers aggregation of a lens protein, betaA3-crystallin

Protein aggregation is a hallmark of several neurodegenerative diseases and also of cataracts. The major proteins in the lens of the eye are crystallins, which accumulate throughout life and are extensively modified. Deamidation is the major modification in the lens during aging and cataracts. Among the crystallins, the betaA3-subunit has been found to have multiple sites of deamidation associated with the insoluble proteins in vivo. Several sites were predicted to be exposed on the surface of betaA3 and were investigated in this study. Deamidation was mimicked by site-directed mutagenesis at Q42 and N54 on the N-terminal domain, N133 and N155 on the C-terminal domain, and N120 in the peptide connecting the domains. Deamidation altered the tertiary structure without disrupting the secondary structure or the dimer formation of betaA3. Deamidations in the C-terminal domain and in the connecting peptide decreased stability to a greater extent than deamidations in the N-terminal domain. Deamidation at N54 and N155 also disrupted the association with the betaB1-subunit. Sedimentation velocity experiments integrated with high-resolution analysis detected soluble aggregates at 15%-20% in all deamidated proteins, but not in wild-type betaA3. These aggregates had elevated frictional ratios, suggesting that they were elongated. The detection of aggregates in vitro strongly suggests that deamidation may contribute to protein aggregation in the lens. A potential mechanism may include decreased stability and/or altered interactions with other beta-subunits. Understanding the role of deamidation in the long-lived crystallins has important implications in other aggregation diseases.     

Laser light-scattering evidence for an altered association of beta B1-crystallin deamidated in the connecting peptide

Deamidation is a prevalent modification of crystallin proteins in the vertebrate lens. The effect of specific sites of deamidation on crystallin stability in vivo is not known. Using mass spectrometry, a previously unreported deamidation in beta B1-crystallin was identified at Gln146. Another deamidation was investigated at Asn157. It was determined that whole soluble beta B1 contained 13%-17% deamidation at Gln146 and Asn157. Static and quasi-elastic laser light scattering, circular dichroism, and heat aggregation studies were used to explore the structure and associative properties of recombinantly expressed wild-type (wt) beta B1 and the deamidated beta B1 mutants, Q146E and N157D. Dimer formation occurred for wt beta B1, Q146E, and N157D in a concentration-dependent manner, but only Q146E showed formation of higher ordered oligomers at the concentrations studied. Deamidation at Gln146, but not Asn157, led to an increased tendency of beta B1 to aggregate upon heating. We conclude that deamidation creates unique effects depending upon where the deamidation is introduced in the crystallin structure.     

The common modification in alphaA-crystallin in the lens, N101D, is associated with increased opacity in a mouse model

To elucidate the morphological and cellular changes due to introduction of a charge during development and the possible mechanism that underlies cataract development in humans as a consequence of an additional charge, we generated a transgenic mouse model mimicking deamidation of Asn at position 101. The mouse model expresses a human αA-crystallin gene in which Asn-101 was replaced with Asp, which is referred to as αAN101D-transgene and is considered to be "deamidated" in this study. Mice expressing αAN101D-transgene are referred to here CRYAA(N101D) mice. All of the lines showed the expression of αAN101D-transgene. Compared with the lenses of mice expressing wild-type (WT) αA-transgene (referred to as CRYAA(WT) mice), the lenses of CRYAA(N101D) mice showed (a) altered αA-crystallin membrane protein (aquaporin-0 (AQP0), a specific lens membrane protein) interaction, (b) extracellular spaces between outer cortical fiber cells, (c) attenuated denucleation during confocal microscopic examination, (d) disrupted normal fiber cell organization and structure during scanning electron microscopic examination, (e) distorted posterior suture lines by bright field microscopy, and (f) development of a mild anterior lens opacity in the superior cortical region during the optical coherence tomography scan analysis. Relative to lenses with WT αA-crystallin, the lenses containing the deamidated αA-crystallin also showed an aggregation of αA-crystallin and a higher level of water-insoluble proteins, suggesting that the morphological and cellular changes in these lenses are due to the N101D mutation. This study provides evidence for the first time that expression of deamidated αA-crystallin caused disruption of fiber cell structural integrity, protein aggregation, insolubilization, and mild cortical lens opacity.     

Structural and functional roles of deamidation and/or truncation of N- or C-termini in human alpha A-crystallin

The purpose of the study was to compare the effects of deamidation alone, truncation alone, or both truncation and deamidation on structural and functional properties of human lens alphaA-crystallin. Specifically, the study investigated whether deamidation of one or two sites in alphaA-crystallin (i.e., alphaA-N101D, alphaA-N123D, alphaA-N101/123D) and/or truncation of the N-terminal domain (residues 1-63) or C-terminal extension (residues 140-173) affected the structural and functional properties relative to wild-type (WT) alphaA. Human WT-alphaA and human deamidated alphaA (alphaA-N101D, alphaA-N123D, alphaA-N101/123D) were used as templates to generate the following eight N-terminal domain (residues 1-63) deleted or C-terminal extension (residues 140-173) deleted alphaA mutants and deamidated plus N-terminal domain or C-terminal extension deleted mutants: (i) alphaA-NT (NT, N-terminal domain deleted), (ii) alphaA-N101D-NT, (iii) alphaA-N123D-NT, (iv) alphaA-N101/123D-NT, (v) alphaA-CT (CT, C-terminal extension deleted), (vi) alphaA-N101D-CT, (vii) alphaA-N123D-CT, and (viii) alphaA-N101/123D-CT. All of the proteins were purified and their structural and functional (chaperone activity) properties determined. The desired deletions in the alphaA-crystallin mutants were confirmed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometric analysis. Relative to WT-alphaA homomers, the mutant proteins exhibited major structural and functional changes. The maximum decrease in chaperone activity in homomers occurred on deamidation of N123 residue, but it was substantially restored after N- or C-terminal truncations in this mutant protein. Far-UV circular dichroism (CD) spectral analyses generally showed an increase in the beta-contents in alphaA mutants with deletions of N-terminal domain or C-terminal extension and also with deamidation plus above N- or C-terminal deletions. Intrinsic tryptophan (Trp) and total fluorescence spectral studies suggested altered microenvironments in the alphaA mutant proteins. Similarly, the ANS (8-anilino-1-naphthalenesulfate) binding showed generally increased fluorescence with blue shift on deletion of the N-terminal domain in the deamidated mutant proteins, but opposite effects were observed on deletion of the C-terminal extension. Molecular mass, polydispersity of homomers, and the rate of subunit exchange with WT-alphaB-crystallin increased on deletion of the C-terminal extension in the deamidated alphaA mutants, but on N-terminal domain deletion these values showed variable results based on the deamidation site. In summary, the data suggested that the deamidation alone showed greater effect on chaperone activity than the deletion of N-terminal domain or C-terminal extension of alphaA-crystallin. The N123 residue of alphaA-crystallin plays a crucial role in maintaining its chaperone function. However, both the N-terminal domain and C-terminal extension are also important for the chaperone activity of alphaA-crystallin because the activity was partially or fully recovered following either deletion in the alphaA-N123D mutant. The results of subunit exchange rates among alphaA mutants and WT-alphaB suggested that such exchange is an important determinant in maintenance of chaperone activity following deamidation and/or deletion of the N-terminal domain or C-terminal extension in alphaA-crystallin.     

Lens β-crystallins: The role of deamidation and related modifications in aging and cataract

Crystallins are the major proteins in the lens of the eye and function to maintain transparency of the lens. Of the human crystallins, α, β, and γ, the β-crystallins remain the most elusive in their structural significance due to their greater number of subunits and possible oligomer formations. The β-crystallins are also heavily modified during aging. This review focuses on the functional significance of deamidation and the related modifications of racemization and isomerization, the major modifications in β-crystallins of the aged human lens. Elucidating the role of these modifications in cataract formation has been slow, because they are analytically among the most difficult post-translational modifications to study. Recent results suggest that many amides deamidate to similar extent in normal aged and cataractous lenses, while others may undergo greater deamidation in cataract. Mimicking deamidation at critical structural regions induces structural changes that disrupt the stability of the β-crystallins and lead to their aggregation in vitro. Deamidations at the surface disrupt interactions with other crystallins. Additionally, the α-crystallin chaperone is unable to completely prevent deamidated β-crystallins from insolubilization. Therefore, deamidation of β-crystallins may enhance their precipitation and light scattering in vivo contributing to cataract formation.     

Glutamine deamidation destabilizes human gammaD-crystallin and lowers the kinetic barrier to unfolding

Human eye lens transparency requires life long stability and solubility of the crystallin proteins. Aged crystallins have high levels of covalent damage, including glutamine deamidation. Human gammaD-crystallin (HgammaD-Crys) is a two-domain beta-sheet protein of the lens nucleus. The two domains interact through interdomain side chain contacts, including Gln-54 and Gln-143, which are critical for stability and folding of the N-terminal domain of HgammaD-Crys. To test the effects of interface deamidation on stability and folding, single and double glutamine to glutamate substitutions were constructed. Equilibrium unfolding/refolding experiments of the proteins were performed in guanidine hydrochloride at pH 7.0, 37 degrees C, or urea at pH 3.0, 20 degrees C. Compared with wild type, the deamidation mutants were destabilized at pH 7.0. The proteins populated a partially unfolded intermediate that likely had a structured C-terminal domain and unstructured N-terminal domain. However, at pH 3.0, equilibrium unfolding transitions of wild type and the deamidation mutants were indistinguishable. In contrast, the double alanine mutant Q54A/Q143A was destabilized at both pH 7.0 and 3.0. Thermal stabilities of the deamidation mutants were also reduced at pH 7.0. Similarly, the deamidation mutants lowered the kinetic barrier to unfolding of the N-terminal domain. These data indicate that interface deamidation decreases the thermodynamic stability of HgammaD-Crys and lowers the kinetic barrier to unfolding due to introduction of a negative charge into the domain interface. Such effects may be significant for cataract formation by inducing protein aggregation or insolubility.     

Association of partially folded lens betaB2-crystallins with the alpha-crystallin molecular chaperone

Age-related cataract is a result of crystallins, the predominant lens proteins, forming light-scattering aggregates. In the low protein turnover environment of the eye lens, the crystallins are susceptible to modifications that can reduce stability, increasing the probability of unfolding and aggregation events occurring. It is hypothesized that the alpha-crystallin molecular chaperone system recognizes and binds these proteins before they can form the light-scattering centres that result in cataract, thus maintaining the long-term transparency of the lens. In the present study, we investigated the unfolding and aggregation of (wild-type) human and calf betaB2-crystallins and the formation of a complex between alpha-crystallin and betaB2-crystallins under destabilizing conditions. Human and calf betaB2-crystallin unfold through a structurally similar pathway, but the increased stability of the C-terminal domain of human betaB2-crystallin relative to calf betaB2-crystallin results in the increased population of a partially folded intermediate during unfolding. This intermediate is aggregation-prone and prevents constructive refolding of human betaB2-crystallin, while calf betaB2-crystallin can refold with high efficiency. alpha-Crystallin can effectively chaperone both human and calf betaB2-crystallins from thermal aggregation, although chaperone-bound betaB2-crystallins are unable to refold once returned to native conditions. Ordered secondary structure is seen to increase in alpha-crystallin with elevated temperatures up to 60 degrees C; structure is rapidly lost at temperatures of 70 degrees C and above. Our experimental results combined with previously reported observations of alpha-crystallin quaternary structure have led us to propose a structural model of how activated alpha-crystallin chaperones unfolded betaB2-crystallin.     

A Rapid,Comprehensive Liquid Chromatography-Mass Spectrometry (LC-MS)-based Survey of the Asp Isomers in Crystallins from Human Cataract Lenses

Cataracts are caused by clouding of the eye lens and may lead to partial or total loss of vision. The mechanism of cataract development, however, is not well understood. It is thought that abnormal aggregates of lens proteins form with age, causing loss of lens clarity and development of the cataract. Lens proteins are composed of soluble α-, β-, and γ-crystallins, and as long lived proteins, they undergo post-translational modifications including isomerization, deamidation, and oxidation, which induce insolubilization, aggregation, and loss of function that may lead to cataracts. Therefore, analysis of post-translational modifications of individual amino acid residues in proteins is important. However, detection of the optical isomers of amino acids formed in these proteins is difficult because optical resolution is only achieved using complex methodology. In this study, we describe a new method for the analysis of isomerization of individual Asp residues in proteins using LC-MS and the corresponding synthetic peptides containing the Asp isomers. This makes it possible to analyze isomers of Asp residues in proteins precisely and quickly. We demonstrate that Asp-58, -76, -84, and -151 of αA-crystallin and Asp-62 and -96 of αB-crystallin are highly converted to lβ-, dβ-, and dα-isomers. The amount of isomerization of Asp is greater in the insoluble fraction at all Asp sites in lens proteins, therefore indicating that isomerization of these Asp residues affects the higher order structure of the proteins and contributes to the increase in aggregation, insolubilization, and disruption of function of proteins in the lens, leading to the cataract.     

A nonsense mutation in CRYBB1 associated with autosomal dominant cataract linked to human chromosome 22q

Autosomal dominant cataract is a clinically and genetically heterogeneous lens disorder that usually presents as a sight-threatening trait in childhood. Here we have mapped dominant pulverulent cataract to the beta-crystallin gene cluster on chromosome 22q11.2. Suggestive evidence of linkage was detected at markers D22S1167 (LOD score [Z] 2.09 at recombination fraction [theta] 0) and D22S1154 (Z=1.39 at theta=0), which closely flank the genes for betaB1-crystallin (CRYBB1) and betaA4-crystallin (CRYBA4). Sequencing failed to detect any nucleotide changes in CRYBA4; however, a G-->T transversion in exon 6 of CRYBB1 was found to cosegregate with cataract in the family. This single-nucleotide change was predicted to introduce a translation stop codon at glycine 220 (G220X). Expression of recombinant human betaB1-crystallin in bacteria showed that the truncated G220X mutant was significantly less soluble than wild type. This study has identified the first CRYBB1 mutation associated with autosomal dominant cataract in humans.     

Reliable detection of deamidated peptides from lens crystallin proteins using changes in reversed-phase elution times and parent ion masses

Identifying deamidated peptides using low-resolution mass spectrometry is difficult because traditional database search programs cannot accurately detect modified peptides when the mass differences are only 0.984 Da. In this study, we utilized differential reversed-phase elution behavior of deamidated and corresponding unmodified peptide forms to significantly improve deamidation detection on a low-resolution LCQ ion trap instrument. We also improved the mass measurements of unmodified and deamidated peptide forms by averaging survey scans across each chromatogram peak. Tryptic digests of a series of normal (3-day old, 2-year old, 18-year old, 35-year old, and 70-year old) and cataractous (93-year old) human lens samples were used to produce large numbers of potentially deamidated peptides. The complex peptide mixtures were separated by strong cation exchange (SCX) chromatography followed by reversed-phase (RP) chromatography. Synthetic peptides were used to show that unmodified and deamidated peptides coeluted during the SCX separation and were completely resolved with the RP conditions used. Retention time shifts (RTS) and mass differences (DeltaM) of deamidated lens peptides and their corresponding unmodified forms were manually determined for the 70-year old lens sample. These values were used to assign correct or incorrect deamidation identifications from SEQUEST searches where deamidation was specified as a variable modification. Manual validation of SEQUEST identifications from synthetic peptides, 3-day old, and 70-year old samples had an overall 42% deamidation detection accuracy. Filtering SEQUEST identifications using RTS and DeltaM constraints resulted in >93% deamidation detection accuracy. An algorithm was developed to automate this method, and 72 Crystallin deamidation sites, 18 of which were not previously reported in human lens tissue, were detected.     

Quantification of the thermodynamically linked quaternary and tertiary structural stabilities of transthyretin and its disease-associated variants: the relationship between stability and amyloidosis

Urea denaturation studies were carried out as a function of transthyretin (TTR) concentration to quantify the thermodynamically linked quaternary and tertiary structural stability and to improve our understanding of the relationship between mutant folding energetics and amyloid disease phenotype. Urea denaturation of TTR involves at least two equilibria: dissociation of tetramers into folded monomers and monomer unfolding. To deal with the thermodynamic linkage of these equilibria, we analyzed concentration-dependent denaturation data by globally fitting them to an equation that simultaneously accounts for the two-step denaturation process. Using this method, the quaternary and tertiary structural stabilities of well-behaved TTR sequences, wild-type (WT) TTR and the disease-associated variant V122I, were scrutinized. The V122I variant is linked to late onset familial amyloid cardiomyopathy, the most common familial TTR amyloid disease. V122I TTR exhibits a destabilized quaternary structure and a stable tertiary structure relative to those of WT TTR. Three other variants of TTR were also examined, L55P, V30M, and A25T TTR. The L55P mutation is associated with the most aggressive familial TTR amyloid disease. L55P TTR has a complicated denaturation pathway that includes dimers and trimers, so globally fitting its concentration-dependent urea denaturation data yielded error-laden estimates of stability parameters. Nevertheless, it is clear that L55P TTR is substantially less stable than WT TTR, primarily because its tertiary structure is unstable, although its quaternary structure is destabilized as well. V30M is the most common mutation associated with neuropathic forms of TTR amyloid disease. V30M TTR is certainly destabilized relative to WT TTR, but like L55P TTR, it has a complex denaturation pathway that cannot be fit to the aforementioned two-step denaturation model. Literature data suggest that V30M TTR has stable quaternary structure but unstable tertiary structure. The A25T mutant, associated with central nervous system amyloidosis, is highly aggregation-prone and exhibits drastically reduced quaternary and tertiary structural stabilities. The observed differences in stability among the disease-associated TTR variants highlight the complexity and heterogeneity of TTR amyloid disease, an observation that has important implications for the treatment of these maladies.     

Site-specific mutagenesis on Mnemiopsin 2: Calcium coordination and substrate binding properties of new variants

We used site-specific mutagenesis by targeting E179 and F190 on the structure of photoprotein Mnemiopsin 2 (Mn2) from Mnemiopsis leidyi. The tertiary structure of E179S and F190L mutants was made by the MODELLER program. Far-ultraviolet circular dichroism data showed that the overall secondary structural content of photoprotein is not changed upon mutation, however the helicity and stabilizing interactions in helical structure decreases in mutants as compared with the wild-type (WT) photoprotein. Fluorescence spectra data revealed that the tertiary structure of the mutants is more compact than that of WT Mn2. According to the heat-induced denaturation experiments data, the melting temperature (T_m) for the unfolding of tertiary structure of the F190L variant increases by 3°C compared with that of the WT and E179S mutant. Interestingly, the conformational enthalpy of the F190L mutant (86 kcal mol⁻¹) is considerably lower than those in the WT photoprotein (102 kcal mol⁻¹) and E179S mutant (106 kcal mol⁻¹). The significant difference in the enthalpy of the thermal unfolding process could be explained by considering that the thermally denatured state of the F190L mutant is structurally less expanded than the WT and E179S variants. Bioluminescence activity data showed that the maximum characteristic wavelengths of the mutants undergo blue shift as compared with the WT protein. Initial intensity of the F190L and E179S variants was recorded to be 137.5% and 55.9% of the WT protein, respectively.     

Structure and stability of gamma-crystallins. Denaturation and proteolysis behavior

The denaturation behavior of bovine lens gamma-crystallin fractions II, III, and IV and their susceptibility to proteolysis in vitro was compared to determine whether differences in their stability could play a role in cataract formation. Tertiary and secondary structure changes induced by increasing concentrations of urea, guanidine hydrochloride, and sodium dodecyl sulfate and by increasingly alkaline pH were followed by near-UV and far-UV circular dichroism, Trp fluorescence emission, and exposure of sulfhydryl groups. Major differences were found in the denaturation and proteolysis behavior of the three gamma-crystallin fractions. In general, the unfolding of gamma-II and gamma-III crystallins is rather gradual, suggesting the presence of intermediate unfolding states; in contrast, the order-disorder transition of gamma-IV crystallin is abrupt. The gamma-IV crystallin fraction is the most stable in urea and guanidine hydrochloride, but is most susceptible to nonspecific proteolysis and alkaline pH denaturation. Differences in denaturation and proteolysis behavior are attributed to the inherent differences in the tertiary structures of these crystallins.     

Identification of an isoaspartyl linkage formed upon deamidation of bovine calbindin D9k and structural characterization by 2D 1H NMR

Preparations of recombinant bovine calbindin D9k (r-calbindin) that appear homogeneous on SDS electrophoresis gels have been shown by isoelectric focusing to be mixtures of proteins differing in net charge. The production of two isoforms with increased negative charge occurs during a routine urea denaturation step and can be effectively suppressed by replacing this procedure with thermal denaturation. The two isoforms have been separated from the native protein by DEAE-Sephacel ion-exchange chromatography. Amino acid sequencing of tryptic peptide fragments and two-dimensional (2D) 1H NMR studies establish that the isoforms correspond to calbindin D9k deamidated at Asn56 and that the major product has an isoaspartate (beta-linked peptide) residue at this position. The minor deamidated component is found to have a normal Asp-Gly alpha-linkage. A detailed analysis of proton chemical shifts, phi backbone dihedral angles, and nuclear Overhauser effects indicates that the global conformation of r-calbindin is not perturbed upon deamidation and that all elements of secondary structure are intact. The Asp56 form is nearly identical with the intact protein, whereas the structure of the iso-Asp56 form is perturbed, predominantly in the polypeptide segment Lys55-Asp58. These studies demonstrate that 2D 1H NMR techniques can be used to identify and quantitate the two isoforms produced upon deamidation of a protein and to assess changes in the local and global conformation.     

Crystal structure of truncated human betaB1-crystallin

Crystallins are long-lived proteins packed inside eye lens fiber cells that are essential in maintaining the transparency and refractive power of the eye lens. Members of the two-domain betagamma-crystallin family assemble into an array of oligomer sizes, forming intricate higher-order networks in the lens cell. Here we describe the 1.4 angstroms resolution crystal structure of a truncated version of human betaB1 that resembles an in vivo age-related truncation. The structure shows that unlike its close homolog, betaB2-crystallin, the homodimer is not domain swapped, but its domains are paired intramolecularly, as in more distantly related monomeric gamma-crystallins. However, the four-domain dimer resembles one half of the crystallographic bovine betaB2 tetramer and is similar to the engineered circular permuted rat betaB2. The crystal structure shows that the truncated betaB1 dimer is extremely well suited to form higher-order lattice interactions using its hydrophobic surface patches, linker regions, and sequence extensions.     

C-terminal lysine truncation increases thermostability and enhances chaperone-like function of porcine alphaB-crystallin

The carboxyl-terminal segment of alpha-crystallin, a major lens protein of all vertebrates, has a short and flexible peptide extension of about 20 amino acid residues that are very susceptible to proteolytic truncation and modifications under physiological conditions. To investigate its role in crystallin aggregation and chaperone-like activity, we constructed a mutant of porcine alphaB-crystallin with C-terminal lysine truncated end, which unexpectedly showed better chaperone-like function than wild-type alphaB-crystallin. From circular dichroism (CD) spectra, we show that the mutant possesses similar secondary and tertiary structures to those of native purified and recombinant alphaB-crystallins. Analytical ultracentrifugation revealed that the truncated mutant was smaller than wild-type alphaB-crystallin in aggregation size and mass. The observed higher thermostability and anti-thermal aggregation propensity of the truncated alphaB-crystallin mutant than wild-type alphaB-crystallin are in contrast to the prevailing notion that mutations at the C-terminal lysines of alphaB-crystallin result in substantial loss of chaperone-like activity, despite the overall preservation of secondary structure. The detailed characterization of the C-terminal deletion mutants may provide some deeper insight into the chaperoning mechanism of the structurally related small heat-shock protein family.     

Site‐specific rapid deamidation and isomerization in human lens αA‐crystallin in vitro

Recent studies have suggested that the isomerization/racemization of aspartate residues in proteins increases in aged tissues. One such residue is Asp151 in lens‐specific αA‐crystallin. Although many isomerization/racemization sites have been reported in various proteins, the factors that lead to those modifications in proteins in vivo remain obscure. Therefore, an in vitro system is needed to assess the mechanisms of modifications of Asp under various conditions. Deamidation of Asn to Asp in proteins occurs more rapidly than isomerization/racemization of Asp, although the reaction passes through the same intermediate in both pathways. Here, therefore, we replaced Asp151 in human lens αA‐crystallin with Asn by using site‐directed mutagenesis. The recombinant protein was expressed in Escherichia coli and used to investigate the deamidation/isomerization/racemization of Asn151 after incubation at 50°C for various durations and under different pH. After incubation, the mutant αA‐crystallin was subjected to enzymatic digestion followed by liquid chromatography–MS/MS to evaluate the ratio of modifications in Asn151‐containing peptides. The Asp151Asn αA‐crystallin mutant showed rapid deamidation to Asp with the formation of specific Asp isomers. In particular, deamidation increased greatly under basic conditions. By contrast, subunit–subunit interactions between αA‐crystallin and αB‐crystallin had little effect on the modification of Asn151. Our findings suggest that the Asp151Asn αA‐crystallin mutant represents a good in vitro model protein to assess deamidation, isomerization, and the racemization intermediates. Furthermore, our in vitro results show a different trend from in vivo data, implying the presence of specific factors that induce racemization from L‐Asp to D‐Asp residues in vivo.