Solution properties of γ‐crystallins: Compact structure and low frictional ratio are conserved properties of diverse γ‐crystallins

γ‐crystallins are highly specialized proteins of the vertebrate eye lens where they survive without turnover under high molecular crowding while maintaining transparency. They share a tightly folded structural template but there are striking differences among species. Their amino acid compositions are unusual. Even in mammals, γ‐crystallins have high contents of sulfur‐containing methionine and cysteine, but this reaches extremes in fish γM‐crystallins with up to 15% Met. In addition, fish γM‐crystallins do not conserve the paired tryptophan residues found in each domain in mammalian γ‐crystallins and in the related β‐crystallins. To gain insight into important, evolutionarily conserved properties and functionality of γ‐crystallins, zebrafish (Danio rerio) γM2b and γM7 were compared with mouse γS and human γD. For all four proteins, far UV CD spectra showed the expected β‐sheet secondary structure. Like the mammalian proteins, γM7 was highly soluble but γM2b was much less so. The heat and denaturant stability of both fish proteins was lower than either mammalian protein. The ability of full‐length and truncated versions of human αB‐crystallin to retard aggregation of the heat denatured proteins also showed differences. However, when solution behavior was investigated by sedimentation velocity experiments, the diverse γ‐crystallins showed remarkably similar hydrodynamic properties with low frictional ratios and partial specific volumes. The solution behavior of γ‐crystallins, with highly compact structures suited for the densely packed environment of the lens, seems to be highly conserved and appears largely independent of amino acid composition.     

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.     

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.     

Membrane dynamics of γ‐secretase with the anterior pharynx‐defective 1B subunit

The four‐subunit protease complex γ‐secretase cleaves many single‐pass transmembrane (TM) substrates, including Notch and β‐amyloid precursor protein to generate amyloid‐β (Aβ), central to Alzheimer's disease. Two of the subunits anterior pharynx‐defective 1 (APH‐1) and presenilin (PS) exist in two homologous forms APH1‐A and APH1‐B, and PS1 and PS2. The consequences of these variations are poorly understood and could affect Aβ production and γ‐secretase medicine. Here, we developed the first complete structural model of the APH‐1B subunit using the published cryo‐electron microscopy (cryo‐EM) structures of APH1‐A (Protein Data Bank: 5FN2, 5A63, and 6IYC). We then performed all‐atom molecular dynamics simulations at 303 K in a realistic bilayer system to understand both APH‐1B alone and in γ‐secretase without and with substrate C83‐bound. We show that APH‐1B adopts a 7TM topology with a water channel topology similar to APH‐1A. We demonstrate direct transport of water through this channel, mainly via Glu84, Arg87, His170, and His196. The apo and holo states closely resemble the experimental cryo‐EM structures with APH‐1A, however with subtle differences: The substrate‐bound APH‐1B γ‐secretase was quite stable, but some TM helices of PS1 and APH‐1B rearranged in the membrane consistent with the disorder seen in the cryo‐EM data. This produces different accessibility of water molecules for the catalytic aspartates of PS1, critical for Aβ production. In particular, we find that the typical distance between the catalytic aspartates of PS1 and the C83 cleavage sites are shorter in APH‐1B, that is, it represents a more closed state, due to interactions with the C‐terminal fragment of PS1. Our structural‐dynamic model of APH‐1B alone and in γ‐secretase suggests generally similar topology but some notable differences in water accessibility which may be relevant to the protein's existence in two forms and their specific function and location.     

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.     

Truncation, cross-linking and interaction of crystallins and intermediate filament proteins in the aging human lens

The optical properties of the lens are dependent upon the integrity of proteins within the fiber cells. During aging, crystallins, the major intra-cellular structural proteins of the lens, aggregate and become water-insoluble. Modifications to crystallins and the lens intermediate filaments have been implicated in this phenomenon. In this study, we examined changes to, and interactions between, human lens crystallins and intermediate filament proteins in lenses from a variety of age groups (0-86years). Among the lens-specific intermediate filament proteins, filensin was extensively cleaved in all postnatal lenses, with truncated products of various sizes being found in both the lens cortical and nuclear extracts. Phakinin was also truncated and was not detected in the lens nucleus. The third major intermediate filament protein, vimentin, remained intact in lens cortical fiber cells across the age range except for an 86year lens, where a single ~49kDa breakdown product was observed. An αB-crystallin fusion protein (maltose-binding protein-αB-crystallin) was found to readily exchange subunits with endogenous α-crystallin, and following mild heat stress, to bind to filensin, phakinin and vimentin and to several of their truncated products. Tryptic digestion of a truncated form of filensin suggested that the binding site for α-crystallin may be in the N-terminal region. The presence of significant amounts of small peptides derived from γS- and βB1-crystallins in the water-insoluble fraction of the lens indicates that these interact tightly with cytoskeletal or membrane components. Interestingly, water-soluble complexes (~40kDa) contained predominantly γS- and βB1-crystallins, suggesting that cross-linking is an alternative pathway for modified β- and γ-crystallins in the lens.     

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.     

Enzyme/crystallins and extremely high pyridine nucleotide levels in the eye lens

Taxon-specific crystallins are proteins present in high abundance in the lens of phylogenetically restricted groups of animals. Recently it has been found that these proteins are actually enzymes which the lens has apparently adopted to serve as structural proteins. Most of these proteins have been shown to be identical to, or related to, oxidoreductases. In guinea pig lens, which contains zeta-crystallin, a protein with an NADPH-dependent oxidoreductase activity, the levels of both NADPH and NADP+ are extremely high and correlate with the concentration of zeta-crystallin. We report here nucleotide assays on lenses from vertebrates containing other enzyme/crystallins. In each case where the enzyme/crystallin is a pyridine nucleotide-binding protein the level of that particular nucleotide is extremely high in the lens. The presence of an enzyme/crystallin does not affect the lenticular concentrations of those nucleotides which are not specifically bound. The possibility that nucleotide binding may be a factor in the selection of some enzymes to serve as enzyme/crystallins is considered.     

Physicochemical characterization of a crystallin from the squid lens and its comparison with vertebrate lens crystallins

A crystallin was isolated from the homogenate of the Squid (Loligo pealii) lens by gel filtration on a Sepharose CL-6B (2.5 X 170 cm) column. Biochemical characterization showed it is a dimeric protein with a molecular weight of (5.1 +/- 0.4) X 10(4) and a Stokes' radius of 26A. Electrophoresis on a cellulose acetate membrane indicated it is a basic protein with an isoelectric point higher than 8.6. High resolution two-dimensional gel in 8 M urea/2% NP-40 resolved this crystallin into 6 charge isomers, each with a major subunit of molecular weight 29,000 daltons and a minor subunit of 27,000 daltons in a molar ratio of 3:1. The extreme susceptibility of the protein to denaturation and precipitation even at low temperature hampered further characterization of this crystallin under nondenaturing conditions. Amino acid analysis indicated it contains an unusually high content of methionine (12.8 mol%) which may have some bearing on the instability of this crystallin in vitro. Biochemical comparison of the squid crystallin with mammalian lens crystallins shows that it is a crystallin distinguishable from all reported vertebrate lens crystallins. A detailed study of this protein may shed light on the evolution of lens crystallins in general.     

The role of macromolecular crowding in the evolution of lens crystallins with high molecular refractive index

Crystallins are present in the lens at extremely high concentrations in order to provide transparency and generate a high refractive power of the lens. The crystallin families prevalent in the highest density lens tissues are γ-crystallins in vertebrates and S-crystallins in cephalopods. As shown elsewhere, in parallel evolution, both have evolved molecular refractive index increments 5-10% above those of most proteins. Although this is a small increase, it is statistically very significant and can be achieved only by very unusual amino acid compositions. In contrast, such a molecular adaptation to aid in the refractive function of the lens did not occur in crystallins that are preferentially located in lower density lens tissues, such as vertebrate α-crystallin and taxon-specific crystallins. In the current work, we apply a model of non-interacting hard spheres to examine the thermodynamic contributions of volume exclusion at lenticular protein concentrations. We show that the small concentration decrease afforded by the higher molecular refractive index increment of crystallins can amplify nonlinearly to produce order of magnitude differences in chemical activities, and lead to reduced osmotic pressure and the reduced propensity for protein aggregation. Quantitatively, this amplification sets in only at protein concentrations as high as those found in hard lenses or the nucleus of soft lenses, in good correspondence to the observed crystallin properties in different tissues and different species. This suggests that volume exclusion effects provide the evolutionary driving force for the unusual refractive properties and the unusual amino acid compositions of γ-crystallins and S-crystallins.     

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.     

The role of the C(2) configuration and methyl substitution on the catalytic activity of novel 2,3,3‐ and 2,7,7‐trimethyl‐substituted γ‐aminonorbornan‐2‐ols

A new set of optically active 2,3,3‐ and 2,7,7‐trimethyl‐substituted γ‐aminonorbornan‐2‐ols have been obtained from 2‐methylenenorbornane‐1‐carbonitriles derived from (+)‐camphor and (?)‐fenchone and probed as chiral ligands for the enantioselective addition of diethylzinc to benzaldehyde. This has allowed the study of the structural factors influencing the chirality transfer, such as variation of the relative configuration at C(2) and steric hindrance at C(2), C(3), and C(7) positions of norbornane, which result in the observance of the important role played by the gem‐dimethyl position in γ‐aminonorbornan‐2‐exo‐ols. An empirical rationalization of the obtained experimental results has been realized on the basis of energetically‐favored diastereomeric Noyori‐like transition states. Chirality 2010. © 2010 Wiley‐Liss, Inc.     

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.     

Physicochemical characterization of γ-crystallins from bovine lens—Hydrodynamic and biochemical properties

A detailed hydrodynamic study has been made on the γ-crystallin of the bovine lens. Sedimentation study indicates that γ-crystallin shows a nearly gaussian peak throughout the course of sedimentation at high speed, using a synthetic boundary cell. The diffusion and sedimentation coefficients are 10.3×10^?7 cm²/sec and 2.51 S, respectively. The weight-average molecular weight of the unfractionated γ-crystallin calculated from sedimentation equilibrium is 21,800. The four major subfractions of γ-crystallin show similar hydrodynamic properties with an intrinsic viscosity of 2.50 ml/g and a Stokes radius of 21 Å. The distinct electrophoretic mobilities exhibited by the four subfractions show gel-concentration dependence and similar slopes in the Ferguson plot, indicative of being charge isomers of the same molecular species. Amino acid analysis of these four subfractions corroborated the conclusions that these γ-crystallin polypeptides are closely related and comprise a multigene family of crystallins. Based on the sedimentation and intrinsic viscosity data, γ-crystallin can be modeled as a prolate ellipsoid with an axial ratio of approximately 3.0 and a hydration factor of 0.27 g water per gram protein. The circular dichroism data for γ-crystallins showed a minimum at about 217 nm, characteristic of a β-sheet conformation. These structural characteristics are in good accord with those derived from X-ray diffraction data for γ-crystallin II.     

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.     

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.     

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.     

Thermodynamic stability of bovine α-crystallin in its interactions with other bovine crystallins

Light scattering measurements were performed on dilute solutions of α-crystallin mixed with different combinations of β_H, β_L and γ-fractions of bovine lens crystallins. Light scattering intensities were obtained as a function of scattering angle, concentration and temperature. The temperature dependence of the second virial coefficients was used to obtain partial molar enthalpy and end entropy of solutions. The difference between the thermodynamic parameters of the crystallin mixtures and those of the weighted averages of the individual components yielded the excess enthalpy and entropy functions of the solutions. Both the excess enthalpy and entropy functions indicated that thermodynamic stability of α-crystallin is progressively enhanced by its interactions with γ(β_H+γ)(β_H+β_L+γ) crystallins. The last two combinations showed negative values both for excess enthalpy as well for excess entropy of solutions. Other combinations demonstrated increasing positive values. This implies that the combination of all four crystallins in the vertebrate lens enables the best solvation property as well as the best packing as opposed to any other single or combinatorial arrangements of crystallins. Similar conclusions have been obtained in the past from water and other vapor sorption studies.     

Structural and mechanistic insights into homocysteine degradation by a mutant of methionine γ‐lyase based on substrate‐assisted catalysis

Methionine γ‐lyse (MGL) catalyzes the α, γ‐elimination of l ‐methionine and its derivatives as well as the α, β‐elimination of l ‐cysteine and its derivatives to produce α‐keto acids, volatile thiols, and ammonia. The reaction mechanism of MGL has been characterized by enzymological studies using several site‐directed mutants. The Pseudomonas putida MGL C116H mutant showed drastically reduced degradation activity toward methionine while retaining activity toward homocysteine. To understand the underlying mechanism and to discern the subtle differences between these substrates, we analyzed the crystal structures of the reaction intermediates. The complex formed between the C116H mutant and methionine demonstrated that a loop structure (Ala51–Asn64) in the adjacent subunit of the catalytic dimer cannot approach the cofactor pyridoxal 5′‐phosphate (PLP) because His116 disrupts the interaction of Asp241 with Lys240, and the liberated side chain of Lys240 causes steric hindrance with this loop. Conversely, in the complex formed between C116H mutant and homocysteine, the thiol moiety of the substrate conjugated with PLP offsets the imidazole ring of His116 via a water molecule, disrupting the interaction of His116 and Asp241 and restoring the interaction of Asp241 with Lys240. These structural data suggest that the Cys116 to His mutation renders the enzyme inactive toward the original substrate, but activity is restored when the substrate is homocysteine due to substrate‐assisted catalysis.