Synergistic Action of Recombinant α-Amylase and Glucoamylase on the Hydrolysis of Starch Granules

Barley α-amylase 1 mutant (AMY) and Lentinula edodes glucoamylase (GLA) were cloned and expressed in Saccharomyces cerevisiae. The purified recombinant AMY hydrolyzed corn and wheat starch granules, respectively, at rates 1.7 and 2.5 times that of GLA under the same reaction conditions. AMY and GLA synergistically enhanced the rate of hydrolysis by ∼3× for corn and wheat starch granules, compared to the sum of the individual activities. The exo-endo synergism did not change by varying the ratio of the two enzymes when the total concentration was kept constant. A yield of 4% conversion was obtained after 25 min 37°C incubation (1 unit total enzyme, 15 mg raw starch granules, pH 5.3). The temperature stability of the enzyme mixtures was ≤50°C, but the initial rate of hydrolysis continued to increase with higher temperatures. Ca⁺⁺ enhanced the stability of the free enzymes at 50°C incubation. Inhibition was observed with the addition of 10 mM Fe⁺⁺ or Cu⁺⁺, while Mg⁺⁺ and EDTA had lesser effect. Reference to a company and/or products is only for purposes of information and does not imply approval of recommendation of the product to the exclusion of others that may also be suitable. All programs and services of the U.S. Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, age, marital status, or handicap.     

Functional Elements in Molecular Chaperone α-Crystallin: Identification of Binding Sites in αB-Crystallin

α-Crystallin, the predominant eye lens protein with sequence homology to small heat shock proteins, acts like a molecular chaperone by suppressing the aggregation of damaged crystallins and proteins. To gain an insight into the amino acid sequences in α-crystallin involved in chaperone-like function, we used a cleavable, fluorescent, photoactive, crosslinking agent, sulfosuccinimidyl-2(7-azido-4-methylcoumarin-3-acetamido)-ethyl-1,3′ dithiopropionate (SAED), to derivatize yeast alcohol dehydrogenase (ADH) and allowed it to complex with bovine α-crystallin at 48°C. The complex was photolyzed and reduced with DTT and the subunits of α-crystallin, αA- and αB-, were separated. Fluorescence analysis showed that both αA- and αB-crystallins interacted with ADH during chaperone-like function. Tryptic digestion, amino acid sequencing, and mass spectral analysis of αB-crystallin revealed that APSWIDTGLSEMR (57-69) and VLGDVIEVHGKHEER (93-107) sequences were involved in binding with ADH.     

Azithromycin Reduces the Production of α-hemolysin and Biofilm Formation in Staphylococcus aureus

Staphylococcus aureus causes a broad range of life-threatening diseases in humans. This bacterium produces a large number of extracellular virulence factors that are closely associated with specific diseases which are controlled by quorum sensing. In this study, we show that azithromycin was active against methicillin-resistant Staphylococcus aureus (MRSA) strains with MICs ranged from 32 to 64 μg/mL. Azithromycin at subinhibitory concentration, markedly reduced the production of α-hemolysin at (1/16MIC, 1/8MIC) and biofilm formation at (1/16MIC, 1/8MIC), respectively. The results indicated that sub-inhibitory concentrations of azithromycin decreased the production of α-hemolysin and biofilm formation in MRSA in a dose-dependent manner. Therefore, azithromycin may be useful in the treatment of α-hemolysin producing and biofilm formation MRSA infections.     

Structural Basis of the Differential Binding of Engineered Knottins to Integrins αVβ3 and α5β1

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A Study of the Binding of Estradiol and 8-Isoestradiol to the Estrogen α-Receptor by Molecular Modeling

The complexes of the estrogen -receptor with estradiol and 8-isoestradiol were comparatively analyzed. The computations of ligand–receptor complexes, carried out using the FLEXX program, allowed us to propose a model for the binding of the analogues of 8-isoestradiol. It was found that rings Cand D of estradiol and 8-isoestradiol are similarly arranged in the ligand-binding pocket and coincide upon the superposition of the corresponding ligand–receptor complexes, whereas rings A and B do not coincide. The oxygen functions in position 17 of the estradiol analogues of both series coincide upon superposition, whereas the phenol 3-hydroxyl groups are 0.05 Å apart. A comparison of the predicted biological properties of modified estradiol analogues of the natural and 8-iso-series with the available experimental data revealed their similarity. Synthetic 2-acetyl analogues of 8-isoestrogens were found to have no uterotropic activity, which is also consistent with the proposed model.     

Carbohydrate and Protein-based Inhibitors of Porcine Pancreatic α-Amylase: Structure Analysis and Comparison of Their Binding Characteristics

The crystal structures of porcine pancreatic α-amylase isozyme II (PPA II) in its free form and complexed with the trestatin A derived pseudo-octasaccharide V-1532 have been determined using Patterson search techniques at resolutions of 2.3 and 2.2 Å, respectively. Seven rings of the competitive inhibitor V-1532 could be detected in the active site region as well as two maltose units in secondary binding sites on the surface.V-1532 occupies the five central sugar binding subsites similar to the PPA/acarbose structure. A sixth ring exists at the reducing end, connecting two symmetry related PPA molecules. The seventh moiety, a 6-hydroxymethylconduritol ring, is located at the non-reducing end. The electron density for this ring is relatively weak, indicating considerable disorder.This study shows that PPA is able to accommodate more than five rings in the active site region, but that additional rings would increase the binding affinity only slightly, which is in accordance with kinetic experiments.A comparison of the structures of free PPA, PPA/V-1532 and PPA/Tendamistat shows the characteristic conformational changes that accompany inhibitor binding and distinguish pseudo-oligosaccharide inhibitors from proteinaceous inhibitors. Although both classes of inhibitors block the sugar binding subsites in the active site region, the extreme specificity and binding affinity of the proteinaceous inhibitors is probably due to an intricate interaction pattern involving areas further away from the catalytic center.     

Molecular Modeling Reveals the Novel Inhibition Mechanism and Binding Mode of Three Natural Compounds to Staphylococcal α-Hemolysin

α-Hemolysin (α-HL) is a self-assembling, channel-forming toxin that is produced as a soluble monomer by Staphylococcus aureus strains. Until now, α-HL has been a significant virulence target for the treatment of S. aureus infection. In our previous report, we demonstrated that some natural compounds could bind to α-HL. Due to the binding of those compounds, the conformational transition of α-HL from the monomer to the oligomer was blocked, which resulted in inhibition of the hemolytic activity of α-HL. However, these results have not indicated how the binding of the α-HL inhibitors influence the conformational transition of the whole protein during the oligomerization process. In this study, we found that three natural compounds, Oroxylin A 7-O-glucuronide (OLG), Oroxin A (ORA), and Oroxin B (ORB), when inhibiting the hemolytic activity of α-HL, could bind to the “stem” region of α-HL. This was completed using conventional Molecular Dynamics (MD) simulations. By interacting with the novel binding sites of α-HL, the ligands could form strong interactions with both sides of the binding cavity. The results of the principal component analysis (PCA) indicated that because of the inhibitors that bind to the “stem” region of α-HL, the conformational transition of α-HL from the monomer to the oligomer was restricted. This caused the inhibition of the hemolytic activity of α-HL. This novel inhibition mechanism has been confirmed by both the steered MD simulations and the experimental data obtained from a deoxycholate-induced oligomerization assay. This study can facilitate the design of new antibacterial drugs against S. aureus.     

Molecular Docking and Dynamic Simulation to Identify α7nAChR Binding Affinity of Flavonoids for the Treatment of Alzheimer's Disease

Background : Alpha-7-nicotinic acetylcholine receptor (α7nAChR), a ligand-gated ion channel is one of the important parts of the cholinergic pathway in the brain and has a remarkable role in Alzheimer's disease (AD). It has been documented that the modulation of α7nAChR with the help of phytoconstituent can be helpful in the treatment of AD. Method : The binding efficacy of fifty flavonoids was evaluated for human α7nAChR using molecular docking. The best two flavonoids shortlisted from docking analysis were then subjected to molecular dynamic simulations for 100 ns to analyze conformational binding stability with the target protein. Further, the druggability of the selected flavonoids was checked using in silico ADMET studies. Result : The top two flavonoids selected based on binding affinity toward the binding site of α7nAChR from molecular docking were amentoflavone (–9.1 kcal/mol) and gallocatechin (–8.8 kcal/mol). The molecular dynamics simulation revealed that amentoflavone and gallocatechin have a stable state during overall simulation time, lesser root mean deviation (RMSD) and root mean square fluctuation (RMSF), and complex of both compounds with protein is stable until 100 ns. Conclusion : The two flavonoids amentoflavone and gallocatechin are potential lead molecules that could be utilized as effective agonists of α7nAChR to combat Alzheimer's disease. Future in vitro and in vivo analyses are required to confirm their effectiveness.     

Positive Selection or Free to Vary? Assessing the Functional Significance of Sequence Change Using Molecular Dynamics

Evolutionary arms races between pathogens and their hosts may be manifested as selection for rapid evolutionary change of key genes, and are sometimes detectable through sequence-level analyses. In the case of protein-coding genes, such analyses frequently predict that specific codons are under positive selection. However, detecting positive selection can be non-trivial, and false positive predictions are a common concern in such analyses. It is therefore helpful to place such predictions within a structural and functional context. Here, we focus on the p19 protein from tombusviruses. P19 is a homodimer that sequesters siRNAs, thereby preventing the host RNAi machinery from shutting down viral infection. Sequence analysis of the p19 gene is complicated by the fact that it is constrained at the sequence level by overprinting of a viral movement protein gene. Using homology modeling, in silico mutation and molecular dynamics simulations, we assess how non-synonymous changes to two residues involved in forming the dimer interface—one invariant, and one predicted to be under positive selection—impact molecular function. Interestingly, we find that both observed variation and potential variation (where a non-synonymous change to p19 would be synonymous for the overprinted movement protein) does not significantly impact protein structure or RNA binding. Consequently, while several methods identify residues at the dimer interface as being under positive selection, MD results suggest they are functionally indistinguishable from a site that is free to vary. Our analyses serve as a caveat to using sequence-level analyses in isolation to detect and assess positive selection, and emphasize the importance of also accounting for how non-synonymous changes impact structure and function.     

Molecular exploration of the α1A-adrenoceptor orthosteric site: Binding site definition for epinephrine,HEAT and prazosin

Despite the physiological and pharmacological importance of the α_1A-adrenoreceptor, the mode of interactions of classical agonists and radioactive ligands with this receptor is not yet clearly defined. Here, we used mutagenesis studies and binding experiments to evaluate the importance of 11 receptor sites for the binding of ¹²⁵I-HEAT, ³H-prazosin and epinephrine. Only one residue (F312) commonly interacts with the three molecules, and, surprisingly, D106 interacts only with epinephrine in a moderate way. Our docking model shows that prazosin and HEAT are almost superimposed into the orthosteric pocket with their tetralone and quinazoline rings close to the phenyl ring of the agonist.     

Sequential Binding of Staphylococcal y-Hemolysin to Human Erythrocytes and Complex Formation of the Hemolysin on the Cell Surface†

Staphylococcal γ-hemolysin consists of two protein components, F (or HγI) and HγII. To elucidate the mode of action of γ-hemolysin, we studied the binding order of F and HγII to human erythrocytes and the cell-bound state of the two components. The binding of F to human erythrocytes preceded the binding of HγII to the cells, and thereafter hemolysis occurred. Western immunoblot analysis of the cell-bound γ-hemolysin indicated that F and HγII components form high-molecular-mass (150–250 kDa) complexes on the erythrocytes. The toxin complexes were recovered in a Triton X-100-insoluble fraction of the erythrocytes, which contains cytoskeleton proteins. Neither the formation of the toxin complex(es) nor hemolysis occurred when the erythrocytes were treated with proteinase K. Abortion of the complex formation on the proteinase K-treated erythrocytes may be due to the failure of the binding of HγII to the cells, because F bound to the proteinase K-treated erythrocytes to the same extent as to the non-treated erythrocytes.     

Binding of γ-crystallin substrate prevents the binding of copper and zinc ions to the molecular chaperone α-crystallin

α-Crystallin is a small heat shock protein and molecular chaperone. Binding of Cu2+ and Zn2+ ions to α-crystallin leads to enhanced chaperone function. Sequestration of Cu2+ by α-crystallin prevents metal-ion mediated oxidation. Here we show that binding of human γD-crystallin (HGD, a natural substrate) to human αA-crystallin (HAA) is inversely related to the binding of Cu2+/Zn2+ ions: The higher the amount of bound HGD, the lower the amount of bound metal ions. Thus, in the aging lens, depletion of free HAA will not only lower chaperone capacity but also lower Cu2+ sequestration, thereby promoting oxidation and cataract.     

Molecular basis for the differential sensitivity of rat and human α9α10 nAChRs to α-conotoxin RgIA

J. Neurochem. (2012) 122, 1137-1144. ABSTRACT: The α9α10 nicotinic acetylcholine receptor (nAChR) may be a potential target in pathophysiology of the auditory system, chronic pain, and breast and lung cancers. Alpha-conotoxins, from the predatory marine snail Conus, are potent nicotinic antagonists, some of which are selective for the α9α10 nAChR. Here, we report a two order of magnitude species difference in the potency of α-conotoxin RgIA for the rat versus human α9α10 nAChR. We investigated the molecular mechanism of this difference. Heterologous expression of the rat α9 with the human α10 subunit in Xenopus oocytes resulted in a receptor that was blocked by RgIA with potency similar to that of the rat α9α10 nAChR. Conversely, expression of the human α9 with that of the rat α10 subunit resulted in a receptor that was blocked by RgIA with potency approaching that of the human α9α10 receptor. Systematic substitution of residues found in the human α9 subunit into the homologous position in the rat α9 subunit revealed that a single point mutation, Thr56 to Ile56, primarily accounts for this species difference. Remarkably, although the α9 nAChR subunit has previously been reported to provide the principal (+) binding face for binding of RgIA, Thr56 is located in the (-) complementary binding face.     

The Structure of the T190M Mutant of Murine α-Dystroglycan at High Resolution: Insight into the Molecular Basis of a Primary Dystroglycanopathy

The severe dystroglycanopathy known as a form of limb-girdle muscular dystrophy (LGMD2P) is an autosomal recessive disease caused by the point mutation T192M in α-dystroglycan. Functional expression analysis in vitro and in vivo indicated that the mutation was responsible for a decrease in posttranslational glycosylation of dystroglycan, eventually interfering with its extracellular-matrix receptor function and laminin binding in skeletal muscle and brain. The X-ray crystal structure of the missense variant T190M of the murine N-terminal domain of α-dystroglycan (50-313) has been determined, and showed an overall topology (Ig-like domain followed by a basket-shaped domain reminiscent of the small subunit ribosomal protein S6) very similar to that of the wild-type structure. The crystallographic analysis revealed a change of the conformation assumed by the highly flexible loop encompassing residues 159–180. Moreover, a solvent shell reorganization around Met190 affects the interaction between the B1–B5 anti-parallel strands forming part of the floor of the basket-shaped domain, with likely repercussions on the folding stability of the protein domain(s) and on the overall molecular flexibility. Chemical denaturation and limited proteolysis experiments point to a decreased stability of the T190M variant with respect to its wild-type counterpart. This mutation may render the entire L-shaped protein architecture less flexible. The overall reduced flexibility and stability may affect the functional properties of α-dystroglycan via negatively influencing its binding behavior to factors needed for dystroglycan maturation, and may lay the molecular basis of the T190M-driven primary dystroglycanopathy.     

Comparative analysis of the sequences of the three collagen chains α1(I), α2 and α1(III): Functional and genetic aspects

The amino acid sequences of type I collagen containing α1(I) and α2 chains at a ratio of 2:1, and of type III collagen consisting of α1 (III) chains are known. A statistical analysis of the sequences of these α chains is presented. The inter-chain comparison showed a high level of homology between the three α chains. The interactive amino acids, such as the polar charged and part of the hydrophobic residues responsible for the assembly of the molecules, are strongly conserved. The intra-chain analysis revealed that the α chains are divided into four related D units, each with a length of 234 residues. Between the D units within a chain the polar residues show a higher variability than the hydrophobic amino acids.Besides the D units, other periodicities such as $\text{D}\text{3}$ (78 residues), $\text{D}\text{6}$ (39 residues), $\text{solD}\text{11}$ (21 residues) and $\text{solD}\text{13}$ (18 residues) were observed, particularly in α1 (I) and α1 (III). The D unit is a functional repeat that is formed by the interactive polar charged and hydrophobic residues and which determines the aggregation of the molecules. The $\text{solD}\text{3}$ unit is mainly pronounced by the non-interactive residues such as proline and alanine and appears to be a reminiscence of a primordial gene. The smaller periodic repeating units may be considered as additional genetic units or as structural units, which determine the triplehelical pitch and thus the lateral aggregation of the molecules.In contrast to α1 (I) and α1 (III), the α2 chain shows less regularity in its internal structure.     

Binding of amphipathic α-helical antimicrobial peptides to lipid membranes: Lessons from temporins B and L

Temporins constitute a family of amphipathic α-helical antimicrobial peptides (AMP) and contain some of the shortest cytotoxic peptides, comprised of only 10-14 residues. General characteristics of temporins parallel those of other AMP, both in terms of structural features and biophysical properties relating to their interactions with membrane lipids, with selective lipid-binding properties believed to underlie the discrimination between target vs host cells. Lipid-binding properties also contribute to the cytotoxicity AMP, causing permeabilization of their target cell membranes. The latter functional property of AMP involves highly interdependent acidic phospholipid-induced conformational changes, aggregation, and formation of toxic oligomers in the membrane. These oligomers are subsequently converted to amyloid-type fibers, as demonstrated for e.g. temporins B and L in our laboratory, and more recently for dermaseptins by Auvynet et al. Amyloid state represents the generic minimum in the folding/aggregation free energy landscape, and for AMP its formation most likely serves to detoxify the peptides, in keeping with the current consensus on mature amyloid being inert and non-toxic. The above scenario is supported by sequence analyses of temporins as well as other amphipathic α-helical AMP belonging to diverse families. Accordingly, sequence comparison identifies ‘conformational switches’, domains with equal probabilities for adopting random coil, α-helical and β-sheet structures. These regions were further predicted also to aggregate and assemble into amyloid β-sheets. Taken together, the lipid-binding properties and structural characterization lend support to the notion that the mechanism of membrane permeabilization by temporins B and L and perhaps of most AMP could be very similar, if not identical, to that of the paradigm amyloid forming cytotoxic peptides, responsible for degenerative cell loss in e.g. prion, Alzheimer's and Parkinson's disease, and type 2 diabetes.     

Binding of the alkaloid aristololactam-β-D-glucoside and daunomycin to human hemoglobin: spectroscopy and calorimetry studies

The interaction of the plant alkaloid aristololactam-β-D-glucoside (ADG) and the anticancer agent daunomycin (DAN) with human hemoglobin was studied by different spectroscopic and calorimetric methods. The binding affinity values of ADG and DAN, estimated from spectroscopic experiments, were 3.79 × 10⁴ and 6.68 × 10⁴ M^?1, respectively. From circular dichroism, 3D fluorescence, and FTIR studies it was observed that, DAN induced stronger conformational changes than ADG in the protein. From synchronous fluorescence spectroscopy results, a pronounced shift in the maximum emission wavelength of tyrosine residues was observed in both cases suggesting that the drugs changed the polarity around tyrosine residues with marginal change around the tryptophan residues. The thermodynamics of the binding interaction analyzed using microcalorimetry presented single binding events that were exothermic in nature in both cases. The binding was driven by large positive standard molar entropy changes with small favorable enthalpy contributions. Negative heat capacity changes in both cases are correlated to the involvement of significant hydrophobic forces in the complexation process. The affinity of DAN to Hb was higher than that of ADG.     

Molecular Mechanics of the α-Actinin Rod Domain: Bending,Torsional, and Extensional Behavior

α-Actinin is an actin crosslinking molecule that can serve as a scaffold and maintain dynamic actin filament networks. As a crosslinker in the stressed cytoskeleton, α-actinin can retain conformation, function, and strength. α-Actinin has an actin binding domain and a calmodulin homology domain separated by a long rod domain. Using molecular dynamics and normal mode analysis, we suggest that the α-actinin rod domain has flexible terminal regions which can twist and extend under mechanical stress, yet has a highly rigid interior region stabilized by aromatic packing within each spectrin repeat, by electrostatic interactions between the spectrin repeats, and by strong salt bridges between its two anti-parallel monomers. By exploring the natural vibrations of the α-actinin rod domain and by conducting bending molecular dynamics simulations we also predict that bending of the rod domain is possible with minimal force. We introduce computational methods for analyzing the torsional strain of molecules using rotating constraints. Molecular dynamics extension of the α-actinin rod is also performed, demonstrating transduction of the unfolding forces across salt bridges to the associated monomer of the α-actinin rod domain.