Solution properties of γ‐crystallins: Hydration of fish and mammal γ‐crystallins

Lens γ crystallins are found at the highest protein concentration of any tissue, ranging from 300 mg/mL in some mammals to over 1000 mg/mL in fish. Such high concentrations are necessary for the refraction of light, but impose extreme requirements for protein stability and solubility. γ‐crystallins, small stable monomeric proteins, are particularly associated with the lowest hydration regions of the lens. Here, we examine the solvation of selected γ‐crystallins from mammals (human γD and mouse γS) and fish (zebrafish γM2b and γM7). The thermodynamic water binding coefficient B₁ could be probed by sucrose expulsion, and the hydrodynamic hydration shell of tightly bound water was probed by translational diffusion and structure‐based hydrodynamic boundary element modeling. While the amount of tightly bound water of human γD was consistent with that of average proteins, the water binding of mouse γS was found to be relatively low. γM2b and γM7 crystallins were found to exhibit extremely low degrees hydration, consistent with their role in the fish lens. γM crystallins have a very high methionine content, in some species up to 15%. Structure‐based modeling of hydration in γM7 crystallin suggests low hydration is associated with the large number of surface methionine residues, likely in adaptation to the extremely high concentration and low hydration environment in fish lenses. Overall, the degree of hydration appears to balance stability and tissue density requirements required to produce and maintain the optical properties of the lens in different vertebrate species.     

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.     

Aggregation of Trp > Glu point mutants of human gamma‐D crystallin provides a model for hereditary or UV‐induced cataract

Numerous mutations and covalent modifications of the highly abundant, long‐lived crystallins of the eye lens cause their aggregation leading to progressive opacification of the lens, cataract. The nature and biochemical mechanisms of the aggregation process are poorly understood, as neither amyloid nor native‐state polymers are commonly found in opaque lenses. The βγ‐crystallin fold contains four highly conserved buried tryptophans, which can be oxidized to more hydrophilic products, such as kynurenine, upon UV‐B irradiation. We mimicked this class of oxidative damage using Trp→Glu point mutants of human γD‐crystallin. Such substitutions may represent a model of UV‐induced photodamage—introduction of a charged group into the hydrophobic core generating “denaturation from within.” The effects of Trp→Glu substitutions were highly position dependent. While each was destabilizing, only the two located in the bottom of the double Greek key fold—W42E and W130E—yielded robust aggregation of partially unfolded intermediates at 37°C and pH 7. The αB‐crystallin chaperone suppressed aggregation of W130E, but not W42E, indicating distinct aggregation pathways from damage in the N‐terminal vs C‐terminal domain. The W130E aggregates had loosely fibrillar morphology, yet were nonamyloid, noncovalent, showed little surface hydrophobicity, and formed at least 20°C below the melting temperature of the native β‐sheets. These features are most consistent with domain‐swapped polymerization. Aggregation of partially destabilized crystallins under physiological conditions, as occurs in this class of point mutants, could provide a simple in vitro model system for drug discovery and optimization.     

β‐strand interactions at the domain interface critical for the stability of human lens γD‐crystallin

Human age‐onset cataracts are believed to be caused by the aggregation of partially unfolded or covalently damaged lens crystallin proteins; however, the exact molecular mechanism remains largely unknown. We have used microseconds of molecular dynamics simulations with explicit solvent to investigate the unfolding process of human lens γD‐crystallin protein and its isolated domains. A partially unfolded folding intermediate of γD‐crystallin is detected in simulations with its C‐terminal domain (C‐td) folded and N‐terminal domain (N‐td) unstructured, in excellent agreement with biochemical experiments. Our simulations strongly indicate that the stability and the folding mechanism of the N‐td are regulated by the interdomain interactions, consistent with experimental observations. A hydrophobic folding core was identified within the C‐td that is comprised of a and b strands from the Greek key motif 4, the one near the domain interface. Detailed analyses reveal a surprising non‐native surface salt‐bridge between Glu135 and Arg142 located at the end of the ab folded hairpin turn playing a critical role in stabilizing the folding core. On the other hand, an in silico single E135A substitution that disrupts this non‐native Glu135‐Arg142 salt‐bridge causes significant destabilization to the folding core of the isolated C‐td, which, in turn, induces unfolding of the N‐td interface. These findings indicate that certain highly conserved charged residues, that is, Glu135 and Arg142, of γD‐crystallin are crucial for stabilizing its hydrophobic domain interface in native conformation, and disruption of charges on the γD‐crystallin surface might lead to unfolding and subsequent aggregation.     

Computationally designed β‐turn foldamers of γ‐peptides based on 2‐(aminomethyl)cyclohexanecarboxylic acid

The γ‐peptide β‐turn structures have been designed computationally by the combination of chirospecific γ 2 , 3 ‐residues of 2‐(aminomethyl)cyclohexanecarboxylic acid (γAmc₆) with a cyclohexyl constraint on the C^α?C^β bond using density functional methods in water. The chirospecific γAmc₆ dipeptide with the (2S,3S)‐(2R,3R) configurations forms a stable turn structure in water, resembling a type II′ turn of α‐peptides, which can be used as a β‐turn motif in β‐hairpins of Ala‐based α‐peptides. The γAmc₆ dipeptide with homochiral (2S,3S)‐(2S,3S) configurations but different cyclohexyl puckerings shows the capability to be incorporated into one of two β‐turn motifs of gramicidin S. The overall structure of this gramicidin S analogue is quite similar to the native gramicidin S with the same patterns and geometries of hydrogen bonds. Our calculated results and the recently observed results may imply the wider applicability of chirospecific γ‐peptides with a cyclohexyl constraint on the backbone to form various peptide foldamers. © 2012 Wiley Periodicals, Inc. Biopolymers 97:1018–1025, 2012.     

The spatial configuration of taxonomic biodiversity along a tropical elevational gradient: α‐, β‐, and γ‐partitions

Biodiversity at larger spatial scales (γ) can be driven by within‐site partitions (α), with little variation in composition among locations, or can be driven by among‐site partitions (β) that signal the importance of spatial heterogeneity. For tropical elevational gradients, we determined the (a) extent to which variation in γ is driven by α‐ or β‐partitions; (b) elevational form of the relationship for each partition; and (c) extent to which elevational gradients are molded by zonation in vegetation or by gradual variation in climatic or abiotic characteristics. We sampled terrestrial gastropods along two transects in the Luquillo Mountains. One passed through multiple vegetation zones (tabonuco, palo colorado, and elfin forests), and one passed through only palm forest. We quantified variation in hierarchical partitions (α, β, and γ) of species richness, evenness, diversity, and dominance, as well as in the content and quality of litter. Total gastropod abundance linearly decreased with increasing elevation along both transects, but was consistently higher in palm than in other forest types. The gradual linear decline in γ‐richness was a consequence of opposing patterns with regard to α‐richness (monotonic decrease) and β‐richness (monotonic increase). For evenness, diversity, and dominance, α‐partitions and γ‐partitions evinced mid‐elevational peaks. The spatial organization of gastropod biodiversity did not mirror the zonation of vegetation. Rather, it was molded by: (a) elevational variation in productivity or nutrient characteristics, (b) the interspersion of palm forest within other forest types, and (c) the cloud condensation point acting as a transition between low and high elevation faunas. Abstract in Spanish is available with online material.     

Structures of single‐layer β‐sheet proteins evolved from β‐hairpin repeats

Free‐standing single‐layer β‐sheets are extremely rare in naturally occurring proteins, even though β‐sheet motifs are ubiquitous. Here we report the crystal structures of three homologous, single‐layer, anti‐parallel β‐sheet proteins, comprised of three or four twisted β‐hairpin repeats. The structures reveal that, in addition to the hydrogen bond network characteristic of β‐sheets, additional hydrophobic interactions mediated by small clusters of residues adjacent to the turns likely play a significant role in the structural stability and compensate for the lack of a compact hydrophobic core. These structures enabled identification of a family of secreted proteins that are broadly distributed in bacteria from the human gut microbiome and are putatively involved in the metabolism of complex carbohydrates. A conserved surface patch, rich in solvent‐exposed tyrosine residues, was identified on the concave surface of the β‐sheet. These new modular single‐layer β‐sheet proteins may serve as a new model system for studying folding and design of β‐rich proteins.     

Conserved buried water molecules enable the β‐trefoil architecture

Available high‐resolution crystal structures for the family of β‐trefoil proteins in the structural databank were queried for buried waters. Such waters were classified as either: (a) unique to a particular domain, family, or superfamily or (b) conserved among all β‐trefoil folds. Three buried waters conserved among all β‐trefoil folds were identified. These waters are related by the threefold rotational pseudosymmetry characteristic of this protein architecture (representing three instances of an identical structural environment within each repeating trefoil‐fold motif). The structural properties of this buried water are remarkable and include: residing in a cavity space no larger than a single water molecule, exhibiting a positional uncertainty (i.e., normalized B‐factor) substantially lower than the average Cα atom, providing essentially ideal H‐bonding geometry with three solvent‐inaccessible main chain groups, simultaneously serving as a bridging H‐bond for three different β‐strands at a point of secondary structure divergence, and orienting conserved hydrophobic side chains to form a nascent core‐packing group. Other published work supports an interpretation that these interactions are key to the formation of an efficient folding nucleus and folded thermostability. The fundamental threefold symmetric structural element of the β‐trefoil fold is therefore, surprisingly, a buried water molecule.     

Molecular modeling reveals binding interface of γ‐tubulin with GCP4 and interactions with noscapinoids

The initiation of microtubule assembly within cells is guided by a cone shaped multi‐protein complex, γ‐tubulin ring complex (γTuRC) containing γ‐tubulin and atleast five other γ‐tubulin‐complex proteins (GCPs), i.e., GCP2, GCP3, GCP4, GCP5, and GCP6. The rim of γTuRC is a ring of γ‐tubulin molecules that interacts, via one of its longitudinal interfaces, with GCP2, GCP3, or GCP4 and, via other interface, with α/β?tubulin dimers recruited for the microtubule lattice formation. These interactions however, are not well understood in the absence of crystal structure of functional reconstitution of γTuRC subunits. In this study, we elucidate the atomic interactions between γ‐tubulin and GCP4 through computational techniques. We simulated two complexes of γ‐tubulin‐GCP4 complex (we called dimer1 and dimer2) for 25 ns to obtain a stable complex and calculated the ensemble average of binding free energies of ?158.82 and ?170.19 kcal/mol for dimer1 and ?79.53 and ?101.50 kcal/mol for dimer2 using MM‐PBSA and MM‐GBSA methods, respectively. These highly favourable binding free energy values points to very robust interactions between GCP4 and γ‐tubulin. From the results of the free‐energy decomposition and the computational alanine scanning calculation, we identified the amino acids crucial for the interaction of γ‐tubulin with GCP4, called hotspots. Furthermore, in the endeavour to identify chemical leads that might interact at the interface of γ‐tubulin‐GCP4 complex; we found a class of compounds based on the plant alkaloid, noscapine that binds with high affinity in a cavity close to γ‐tubulin‐GCP4 interface compared with previously reported compounds. All noscapinoids displayed stable interaction throughout the simulation, however, most robust interaction was observed for bromo‐noscapine followed by noscapine and amino‐noscapine. This offers a novel chemical scaffold for γ‐tubulin binding drugs near γ‐tubulin‐GCP4 interface. Proteins 2015; 83:827–843. © 2015 Wiley Periodicals, Inc.     

β‐Bulges: Extensive structural analyses of β‐sheets irregularities

β‐Sheets are quite frequent in protein structures and are stabilized by regular main‐chain hydrogen bond patterns. Irregularities in β‐sheets, named β‐bulges, are distorted regions between two consecutive hydrogen bonds. They disrupt the classical alternation of side chain direction and can alter the directionality of β‐strands. They are implicated in protein‐protein interactions and are introduced to avoid β‐strand aggregation. Five different types of β‐bulges are defined. Previous studies on β‐bulges were performed on a limited number of protein structures or one specific family. These studies evoked a potential conservation during evolution. In this work, we analyze the β‐bulge distribution and conservation in terms of local backbone conformations and amino acid composition. Our dataset consists of 66 times more β‐bulges than the last systematic study (Chan et al. Protein Science 1993, 2:1574–1590). Novel amino acid preferences are underlined and local structure conformations are highlighted by the use of a structural alphabet. We observed that β‐bulges are preferably localized at the N‐ and C‐termini of β‐strands, but contrary to the earlier studies, no significant conservation of β‐bulges was observed among structural homologues. Displacement of β‐bulges along the sequence was also investigated by Molecular Dynamics simulations.     

γ‐Tubulin complex in Trypanosoma brucei: molecular composition,subunit interdependence and requirement for axonemal central pair protein assembly

γ‐Tubulin complex constitutes a key component of the microtubule‐organizing center and nucleates microtubule assembly. This complex differs in complexity in different organisms: the budding yeast contains the γ‐tubulin small complex (γTuSC) composed of γ‐tubulin, gamma‐tubulin complex protein (GCP)2 and GCP3, whereas animals contain the γ‐tubulin ring complex (γTuRC) composed of γTuSC and three additional proteins, GCP4, GCP5 and GCP6. In Trypanosoma brucei, the composition of the γ‐tubulin complex remains elusive, and it is not known whether it also regulates assembly of the subpellicular microtubules and the spindle microtubules. Here we report that the γ‐tubulin complex in T. brucei is composed of γ‐tubulin and three GCP proteins, GCP2‐GCP4, and is primarily localized in the basal body throughout the cell cycle. Depletion of GCP2 and GCP3, but not GCP4, disrupted the axonemal central pair microtubules, but not the subpellicular microtubules and the spindle microtubules. Furthermore, we showed that the γTuSC is required for assembly of two central pair proteins and that γTuSC subunits are mutually required for stability. Together, these results identified an unusual γ‐tubulin complex in T. brucei, uncovered an essential role of γTuSC in central pair protein assembly, and demonstrated the interdependence of individual γTuSC components for maintaining a stable complex.     

Group II archaeal chaperonin recognition of partially folded human γD‐crystallin mutants

The features in partially folded intermediates that allow the group II chaperonins to distinguish partially folded from native states remain unclear. The archaeal group II chaperonin from Methanococcus Mauripaludis (Mm‐Cpn) assists the in vitro refolding of the well‐characterized β‐sheet lens protein human γD‐crystallin (HγD‐Crys). The domain interface and buried cores of this Greek key conformation include side chains, which might be exposed in partially folded intermediates. We sought to assess whether particular features buried in the native state, but absent from the native protein surface, might serve as recognition signals. The features tested were (a) paired aromatic side chains, (b) side chains in the interface between the duplicated domains of HγD‐Crys, and (c) side chains in the buried core which result in congenital cataract when substituted. We tested the Mm‐Cpn suppression of aggregation of these HγD‐Crys mutants upon dilution out of denaturant. Mm‐Cpn was capable of suppressing the off‐pathway aggregation of the three classes of mutants indicating that the buried residues were not recognition signals. In fact, Mm‐Cpn recognized the HγD‐Crys mutants better than (wild‐type) WT and refolded most mutant HγD‐Crys to levels twice that of WT HγD‐Crys. This presumably represents the increased population or longer lifetimes of the partially folded intermediates of the mutant proteins. The results suggest that Mm‐Cpn does not recognize the features of HγD‐Crys tested—paired aromatics, exposed domain interface, or destabilized core—but rather recognizes other features of the partially folded β‐sheet conformation that are absent or inaccessible in the native state of HγD‐Crys.     

Lens proteomics: analysis of rat crystallins when lenses are exposed to dexamethasone

To identify glucocorticoid induced cataract (GIC)-specific modified crystallins and related changes, we analyzed rat crystallins and related changes in lenses exposed to dexamethasone (Dex). To carry out proteomics analyses, we separated soluble lens proteins with two-dimensional electrophoresis (2-DE) and modified crystallins were analyzed with matrix assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF-MS/MS). Related changes in mRNA, protein levels and morphological and functional changes of modified crystallins were also determined. Measured masses (except for γD-crystallin as the larger and cross-link form), the isoelectric points (PIs; except for βB3-crystallin as the alkalinization form) and amino acid sequences of all known rat crystallins matched previously reported data. Analysis by 2-DE indicated that αA, αB, βB3 and γD increased when lenses were exposed to 5 μM Dex; βA4 increased when lenses were exposed to 1 μM Dex and the five proteins that had the highest expressional trend were identical with the results of Q-PCR. βA3/A1 crystallin (expressional trend identical with results of Q-PCR) and the serum albumin precursor gradually disappeared when exposed to 1-50 μM Dex. Results of Western blotting, immunohistochemistry or fluorescence analysis showed that αA and αB increased most when exposed to 5 μM Dex and βA1/A3 and KI-67 decreased obviously when exposed to 1-50 μM Dex. Electron microscopy showed that the condition of the lens was better when lenses were exposed to 5 μM Dex than at other levels and cracks between the fiber cells became larger when lenses were exposed to 1-50 μM Dex. A chaperone role of α-crystallin protecting heated catalase (CAT) and the activity of superoxide dismutase (SOD), glutathione (GSH), and caspase-3 were highest when exposed to 5 μM Dex. Moreover, αA-crystallins were associated with increased phosphorylation (PI decreased). In conclusion, the proteomics analysis and related changes of rat crystallins when lenses were exposed to Dex in this study will be useful for comparison with normal lens proteins and GIC. We also provided a mechanism for GIC from a proteomics aspect based on the in vitro model.     

Denaturation induced aggregation in α‐crystallin: differential action of chaotropes

α‐Crystallin is a member of small heat shock proteins and is believed to play an exceptional role in the stability of eye lens proteins. The disruption or denaturation of the protein arrangement or solubility of the crystallin proteins can lead to vision problems including cataract. In the present study, we have examined the effect of chemical denaturants urea and guanidine hydrochloride (GdnHCl) on α‐crystallin aggregation, with special emphasis on protein conformational changes, unfolding, and amyloid fibril formation. GdnHCl (4 M) induced a 16 nm red shift in the intrinsic fluorescence of α‐crystallin, compared with 4 nm shift by 8 M urea suggesting a major change in α‐crystallin structure. Circular dichroism analysis showed marked increase in the ellipticity of α‐crystallin at 216 nm, suggesting gain in β‐sheet structure in the presence of GdnHCl (0.5–1 M) followed by unfolding at higher concentration (2–6 M). However, only minor changes in the secondary structure of α‐crystallin were observed in the presence of urea. Moreover, 8‐anilinonaphthalene‐1‐sulfonic acid fluorescence measurement in the presence of GdnHCl and urea showed changes in the hydrophobicity of α‐crystallin. Amyloid studies using thioflavin T fluorescence and congo red absorbance showed that GdnHCl induced amyloid formation in α‐crystallin, whereas urea induced aggregation in this protein. Electron microscopy studies further confirmed amyloid formation of α‐crystallin in the presence of GdnHCl, whereas only aggregate‐like structures were observed in α‐crystallin treated with urea. Our results suggest that α‐crystallin is susceptible to unfolding in the presence of chaotropic agents like urea and GdnHCl. The destabilized protein has increased likelihood to fibrillate. Copyright © 2016 John Wiley & Sons, Ltd.     

Evolution of crystallins for a role in the vertebrate eye lens

The camera eye lens of vertebrates is a classic example of the re‐engineering of existing protein components to fashion a new device. The bulk of the lens is formed from proteins belonging to two superfamilies, the α ‐crystallins and the β γ ‐crystallins. Tracing their ancestry may throw light on the origin of the optics of the lens. The α ‐crystallins belong to the ubiquitous small heat shock proteins family that plays a protective role in cellular homeostasis. They form enormous polydisperse oligomers that challenge modern biophysical methods to uncover the molecular basis of their assembly structure and chaperone‐like protein binding function. It is argued that a molecular phenotype of a dynamic assembly suits a chaperone function as well as a structural role in the eye lens where the constraint of preventing protein condensation is paramount. The main cellular partners of α ‐crystallins, the β ‐ and γ ‐crystallins, have largely been lost from the animal kingdom but the superfamily is hugely expanded in the vertebrate eye lens. Their structures show how a simple Greek key motif can evolve rapidly to form a complex array of monomers and oligomers. Apart from remaining transparent, a major role of the partnership of α ‐crystallins with β ‐ and γ ‐crystallins in the lens is to form a refractive index gradient. Here, we show some of the structural and genetic features of these two protein superfamilies that enable the rapid creation of different assembly states, to match the rapidly changing optical needs among the various vertebrates.     

The hybrid Four‐CBS‐Domain KINβγ subunit functions as the canonical γ subunit of the plant energy sensor SnRK1

The AMPK/SNF1/SnRK1 protein kinases are a family of ancient and highly conserved eukaryotic energy sensors that function as heterotrimeric complexes. These typically comprise catalytic α subunits and regulatory β and γ subunits, the latter function as the energy‐sensing modules of animal AMPK through adenosine nucleotide binding. The ability to monitor accurately and adapt to changing environmental conditions and energy supply is essential for optimal plant growth and survival, but mechanistic insight in the plant SnRK1 function is still limited. In addition to a family of γ‐like proteins, plants also encode a hybrid βγ protein that combines the Four‐Cystathionine β‐synthase (CBS)‐domain (FCD) structure in γ subunits with a glycogen‐binding domain (GBD), typically found in β subunits. We used integrated functional analyses by ectopic SnRK1 complex reconstitution, yeast mutant complementation, in‐depth phylogenetic reconstruction, and a seedling starvation assay to show that only the hybrid KINβγ protein that recruited the GBD around the emergence of the green chloroplast‐containing plants, acts as the canonical γ subunit required for heterotrimeric complex formation. Mutagenesis and truncation analysis further show that complex interaction in plant cells and γ subunit function in yeast depend on both a highly conserved FCD and a pre‐CBS domain, but not the GBD. In addition to novel insight into canonical AMPK/SNF/SnRK1 γ subunit function, regulation and evolution, we provide a new classification of plant FCD genes as a convenient and reliable tool to predict regulatory partners for the SnRK1 energy sensor and novel FCD gene functions.     

Protein accumulation and rumen stability of wheat γ‐gliadin fusion proteins in tobacco and alfalfa

The nutritional value of various crops can be improved by engineering plants to produce high levels of proteins. For example, because methionine deficiency limits the protein quality of Medicago Sativa (alfalfa) forage, producing alfalfa plants that accumulate high levels of a methionine‐rich protein could increase the nutritional value of that crop. We used three strategies in designing methionine‐rich recombinant proteins that could accumulate to high levels in plants and thereby serve as candidates for improving the protein quality of alfalfa forage. In tobacco, two fusion proteins, γ‐gliadin‐δ‐zein and γ‐δ‐zein, as well as δ‐zein co‐expressed with β‐zein, all formed protein bodies. However, the γ‐gliadin‐δ‐zein fusion protein accumulated to the highest level, representing up to 1.5% of total soluble protein (TSP) in one transformant. In alfalfa, γ‐gliadin‐δ‐zein accumulated to 0.2% of TSP, and in an in vitro rumen digestion assay, γ‐gliadin‐δ‐zein was more resistant to microbial degradation than Rubisco. Additionally, although it did not form protein bodies, a γ‐gliadin‐GFP fusion protein accumulated to much higher levels, 7% of TSP, than a recombinant protein comprised of an ER localization signal fused to GFP in tobacco. Based on our results, we conclude that γ‐gliadin‐δ‐zein is a potential candidate protein to use for enhancing methionine levels in plants and for improving rumen stability of forage protein. γ‐gliadin fusion proteins may provide a general platform for increasing the accumulation of recombinant proteins in transgenic plants.     

Functions of crystallins in and out of lens: Roles in elongated and post-mitotic cells

The vertebrate lens evolved to collect light and focus it onto the retina. In development, the lens grows through massive elongation of epithelial cells possibly recapitulating the evolutionary origins of the lens. The refractive index of the lens is largely dependent on high concentrations of soluble proteins called crystallins. All vertebrate lenses share a common set of crystallins from two superfamilies (although other lineage specific crystallins exist). The α-crystallins are small heat shock proteins while the β- and γ-crystallins belong to a superfamily that contains structural proteins of uncertain function. The crystallins are expressed at very high levels in lens but are also found at lower levels in other cells, particularly in retina and brain. All these proteins have plausible connections to maintenance of cytoplasmic order and chaperoning of the complex molecular machines involved in the architecture and function of cells, particularly elongated and post-mitotic cells. They may represent a suite of proteins that help maintain homeostasis in such cells that are at risk from stress or from the accumulated insults of aging.     

Crystallin distribution patterns in concentric layers from toad eye lenses

Protein distribution patterns across eye lenses from the Asiatic toad Bufo gargarizans were investigated and individual crystallin classes characterised. Special fractionation that follows the growth mode of the lens was used to yield nine fractions corresponding to layers laid down at different chronological (developmental) stages. Proportions of soluble and insoluble crystallins within each fraction were measured by Bradford assay. Water‐soluble proteins in all fractions were separated by size‐exclusion HPLC and constituents of each class further characterised by electrophoresis, RP‐HPLC and MS analysis. In outer lens layers, α‐crystallin is the most abundant soluble protein but is not found in soluble proteins in the lens centre. Water‐soluble β‐crystallins also decrease from their highest level in the outer lens to negligible mounts in the central lens. The proportion of soluble γ‐crystallin increases significantly towards the lens centre where this is the only soluble protein present. Insoluble protein levels increase significantly towards the lens centre. In B. gargarizans lenses, as with other anurans, the predominant water‐soluble protein class is γ‐crystallin. No taxon‐specific crystallins were found. The relationship between the protein distribution patterns and the functional properties of the lens this species is discussed.     

Biochemical characterization of crystallins from frog lenses

Lens crystallins were isolated from the homogenate of frog (Rana catesbeiana) eye lenses by gel permeation chromatography and characterized by gel electrophoresis, amino acid analysis and circular dichroism. Four well-defined fractions corresponding to alpha/beta-, beta-, frog 39.5 kDa and gamma-crystallins comprising the relative weight percentages in the total soluble cytoplasmic proteins of 18%, 15%, 14% and 48% respectively were obtained. The native molecular masses for each purified fraction were determined to be 432, 207, 40 and 23 kDa, respectively. The polypeptide compositions as determined by SDS-gel electrophoresis revealed the typical subunit structures of mammalian crystallins with the exception of 39.5 kDa monomeric crystallin, which has not been shown in other classes of vertebrate lenses. The spectra of circular dichroism indicate a predominant beta-sheet structure in all four fractions, which also bears a resemblance to the secondary structure of mammalian crystallins. Comparison of the amino acid compositions of frog crystallins with those of mammalian and fish crystallins suggests that gamma-crystallin from the frog is more closely related to that of porcine than fish crystallins, and the frog 39.5 kDa, frog beta- and lamprey 48 kDa crystallins are probably mutually interrelated.