Crystallization and X-ray diffraction data analysis of oxyhemoglobin-I from the catfish Liposarcus anisitsi (pisces)

Hemoglobin remains, despite the enormous amount of research involving this molecule, as a prototype for allosteric models and new conformations. Functional studies carried out on Hemoglobin-I from the South-American Catfish Liposarcus anisitsi [1] suggest the existence of conformational states beyond those already described for human hemoglobin, which could be confirmed crystallographically. The present work represents the initial steps towards that goal.

Structure of tick anticoagulant peptide at 1.6 Å resolution complexed with bovine pancreatic trypsin inhibitor

The structure of tick anticoagulant peptide (TAP) has been determined by X-ray crystallography at t.6 Å resolution complexed with bovine pancreatic trypsin inhibitor (BPTI). The TAP-BPTI crystals are tetragonal, a = b = 46.87, c = 50.35 Å, space group P41, four complexes per unit cell. The TAP molecules are highly dipolar and form an intermolecular helical array along the c-axis with a diameter of about 45 Å. Individual TAP units interact in a head-to-tail fashion, the positive end of one molecule associating with the distal negative end of another, and vice versa. The BPTI molecules have a uniformly distributed positively charged surface that interacts extensively through 14 hydrogen bonds and two hydrogen bonded salt bridges with the helical groove around the helical TAP chains. Comparing the structure of TAP in TAP-BPTI with TAP bound to factor Xa(Xa) suggests a massive reorganization in the N-terminal tetrapeptide and the first disulfide loop of TAP (CyS5(T)- Cys 15(T)) upon binding to Xa. The Tyr1(T)OH atom of TAP moves 14.2 Å to interact with Asp189 of the S1 specificity site, Arg3(T)CZ moves 5.0 Å with the guanidinium group forming a cation-π-electron complex in the S4 subsite of Xa, while Lys7(T)NZ differs in position by 10.6 Å in TAP-BPTI and TAP-Xa, all of which indicates a different pre-Xa-bound conformation for the N- terminal of TAP in its native state. In contrast to TAP, the BPTI structure of TAP-BPTI is practically the same as all those of previously determined structures of BPTI, only arginine and lysine side-chain conformations showing significant differences.

Coil to extended coil transition in polygalacturonic acid: Conductometric titration and monte carlo simulations

The conformational transition from coil to extended coil for polygalacturonic acid has been studied by conductometric titrations and Monte Carlo simulations. The results of conductometric titrations at different polymer concentrations have been analyzed using the model proposed by Manning,1 which describes the conductivity of polyelectrolitic solutions. This experimental approach provides the transport factor and the average distance between charged groups at different degrees of ionization (α). The mean distances between charged groups have been compared with the values obtained by Monte Carlo simulations. In these simulations the polymer chain is modeled as a self-avoiding random walk in a cubic lattice. The monomers interact through the unscreened Coulombic potential. The ratio between the end-to-end distance and the number of ionized beads provides the average distance between charged monomers. The experimental and theoretical values are in good agreement for the whole range of ionization degrees accessed by conductometric titrations. These results suggest that the electrostatic interactions seem to be the major contribution for the coil to extended coil conformational change. The small deviations for α ≤ 0.5 suggests that the stiffness of the chain, associated with local interactions, becomes increasingly significant as the fraction of charged groups is decreased. © 2000 American Chemical Society.

Biophysical Characterization of Interactions Involving Importin-α during Nuclear Import

Proteins containing the classical nuclear localization sequences (NLSs) are imported into the nucleus by the importin-α/β heterodimer. Importin-α contains the NLS binding site, whereas importin-β mediates the translocation through the nuclear pore. We characterized the interactions involving importin-α during nuclear import using a combination of biophysical techniques (biosensor, crystallography, sedimentation equilibrium, electrophoresis, and circular dichroism). Importin-α is shown to exist in a monomeric autoinhibited state (association with NLSs undetectable by biosensor). Association with importin-β (stoichiometry, 1:1; K D = 1.1 × 10 -8 M) increases the affinity for NLSs; the importin-α/β complex binds representative monopartite NLS (simian virus 40 large T-antigen) and bipartite NLS (nucleoplasmin) with affinities (K D = 3.5 × 10 -8 M and 4.8 × 10 -8 M, respectively) comparable with those of a truncated importin-α lacking the autoinhibitory domain (T-antigen NLS, K D = 1.7 × 10 -8 M; nucleoplasmin NLS, K D = 1.4 × 10 -8 M). The autoinhibitory domain (as a separate peptide) binds the truncated importin-α, and the crystal structure of the complex resembles the structure of full-length importin-α. Our results support the model of regulation of nuclear import mediated by the intrasteric autoregulatory sequence of importin-α and provide a quantitative description of the binding and regulatory steps during nuclear import.

Kinetics and crystal structure of human purine nucleoside phosphorylase in complex with 7-methyl-6-thio-guanosine

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of nucleosides and deoxynucleosides, generating ribose 1-phosphate and the purine base, which is an important step of purine catabolism pathway. The lack of such an activity in humans, owing to a genetic disorder, causes T-cell impairment, and drugs that inhibit this enzyme may have the potential of being utilized as modulators of the immunological system to treat leukemia, autoimmune diseases, and rejection in organ transplantation. Here, we describe kinetics and crystal structure of human PNP in complex with 7-methyl-6-thio-guanosine, a synthetic substrate, which is largely used in activity assays. Analysis of the structure identifies different protein conformational changes upon ligand binding, and comparison of kinetic and structural data permits an understanding of the effects of atomic substitution on key positions of the synthetic substrate and their consequences to enzyme binding and catalysis. Such knowledge may be helpful in designing new PNP inhibitors. © 2005 Elsevier Inc. All rights reserved.

Inactivation of vesicular stomatitis virus through inhibition of membrane fusion by chemical modification of the viral glycoprotein

Membrane fusion is an essential step in the entry of enveloped viruses into their host cells triggered by conformational changes in viral glycoproteins. We have demonstrated previously that modification of vesicular stomatitis virus (VSV) with diethylpyrocarbonate (DEPC) abolished conformational changes on VSV glycoprotein and the fusion reaction catalyzed by the virus. In the present study, we evaluated whether treatment with DEPC was able to inactivate the virus. Infectivity and viral replication were abolished by viral treatment with 0.5 mM DEPC. Mortality profile and inflammatory response in the central nervous system indicated that G protein modification with DEPC eliminates the ability of the virus to cause disease. In addition, DEPC treatment did not alter the conformational integrity of surface proteins of inactivated VSV as demonstrated by transmission electron microscopy and competitive ELISA. Taken together, our results suggest a potential use of histidine (His) modification to the development of a new process of viral inactivation based on fusion inhibition. © 2006 Elsevier B.V. All rights reserved.

The inhibition of 5-enolpyruvylshikimate-3-phosphate synthase as a model for development of novel antimicrobials

EPSP synthase (EPSPS) is an essential enzyme in the shikimate pathway, transferring the enolpyruvyl group of phosphoenolpyruvate to shikimate-3-phosphate to form 5-enolpyruvyl-3-shikimate phosphate and inorganic phosphate. This enzyme is composed of two domains, which are formed by three copies of βαβαββ-folding units; in between there are two crossover chain segments hinging the nearly topologically symmetrical domains together and allowing conformational changes necessary for substrate conversion. The reaction is ordered with shikimate-3-phosphate binding first, followed by phosphoenolpyruvate, and then by the subsequent release of phosphate and EPSP. N-[phosphomethyl]glycine (glyphosate) is the commercial inhibitor of this enzyme. Apparently, the binding of shikimate-3-phosphate is necessary for glyphosate binding, since it induces the closure of the two domains to form the active site in the interdomain cleft. However, it is somehow controversial whether binding of shikimate-3-phosphate alone is enough to induce the complete conversion to the closed state. The phosphoenolpyruvate binding site seems to be located mainly on the C-terminal domain, while the binding site of shikimate-3-phosphate is located primarily in the N-terminal domain residues. However, recent results demonstrate that the active site of the enzyme undergoes structural changes upon inhibitor binding on a scale that cannot be predicted by conventional computational methods. Studies of molecular docking based on the interaction of known EPSPS structures with (R)- phosphonate TI analogue reveal that more experimental data on the structure and dynamics of various EPSPS-ligand complexes are needed to more effectively apply structure-based drug design of this enzyme in the future. © 2007 Bentham Science Publishers Ltd.

Analysis of agonist and antagonist effects on thyroid hormone receptor conformation by hydrogen/deuterium exchange

Thyroid hormone receptors (TRs) are ligand-gated transcription factors with critical roles in development and metabolism. Although x-ray structures of TR ligand-binding domains (LBDs) with agonists are available, comparable structures without ligand (apo-TR) or with antagonists are not. It remains important to understand apo-LBD conformation and the way that it rearranges with ligands to develop better TR pharmaceuticals. In this study, we conducted hydrogen/deuterium exchange on TR LBDs with or without agonist (T 3) or antagonist (NH3). Both ligands reduce deuterium incorporation into LBD amide hydrogens, implying tighter overall folding of the domain. As predicted, mass spectroscopic analysis of individual proteolytic peptides after hydrogen/ deuterium exchange reveals that ligand increases the degree of solvent protection of regions close to the buried ligand-binding pocket. However, there is also extensive ligand protection of other regions, including the dimer surface at H10-H11, providing evidence for allosteric communication between the ligand-binding pocket and distant interaction surfaces. Surprisingly, Cterminal activation helix H12, which is known to alter position with ligand, remains relatively protected from solvent in all conditions suggesting that it is packed against the LBD irrespective of the presence or type of ligand. T 3, but not NH3, increases accessibility of the upper part of H3-H5 to solvent, and we propose that TR H12 interacts with this region in apo-TR and that this interaction is blocked by T 3 but not NH3.Wepresent data from site-directed mutagenesis experiments and molecular dynamics simulations that lend support to this structural model of apo-TR and its ligand-dependent conformational changes. (Molecular Endocrinology 25: 15-31, 2011). Copyright © 2011 by The Endocrine Society.

Structural insights into selectivity and cofactor binding in snake venom l-amino acid oxidases

l-Amino acid oxidases (LAAOs) are flavoenzymes that catalytically deaminate l-amino acids to corresponding α-keto acids with the concomitant production of ammonia (NH 3) and hydrogen peroxide (H 2O 2). Particularly, snake venom LAAOs have been attracted much attention due to their diverse clinical and biological effects, interfering on human coagulation factors and being cytotoxic against some pathogenic bacteria and Leishmania ssp. In this work, a new LAAO from Bothrops jararacussu venom (BjsuLAAO) was purified, functionally characterized and its structure determined by X-ray crystallography at 3.1å resolution. BjsuLAAO showed high catalytic specificity for aromatic and aliphatic large side-chain amino acids. Comparative structural analysis with prokaryotic LAAOs, which exhibit low specificity, indicates the importance of the active-site volume in modulating enzyme selectivity. Surprisingly, the flavin adenine dinucleotide (FAD) cofactor was found in a different orientation canonically described for both prokaryotic and eukaryotic LAAOs. In this new conformational state, the adenosyl group is flipped towards the 62-71 loop, being stabilized by several hydrogen-bond interactions, which is equally stable to the classical binding mode. © 2012 Elsevier Inc.

Hyaluronidase from the venom of the social wasp Polybia paulista (Hymenoptera, Vespidae): Cloning, structural modeling, purification, and immunological analysis

In this study, we describe the cDNA cloning, sequencing, and 3-D structure of the allergen hyaluronidase from Polybia paulista venom (Pp-Hyal). Using a proteomic approach, the native form of Pp-Hyal was purified to homogeneity and used to produce a Pp-specific polyclonal antibody. The results revealed that Pp-Hyal can be classified as a glycosyl hydrolase and that the full-length Pp-Hyal cDNA (1315 bp; GI: 302201582) is similar (80-90%) to hyaluronidase from the venoms of endemic Northern wasp species. The isolated mature protein is comprised of 338 amino acids, with a theoretical pI of 8.77 and a molecular mass of 39,648.8 Da versus a pI of 8.13 and 43,277.0 Da indicated by MS. The Pp-Hyal 3D-structural model revealed a central core (α/β)7 barrel, two sulfide bonds (Cys 19-308 and Cys 185-197), and three putative glycosylation sites (Asn79, Asn187, and Asn325), two of which are also found in the rVes v 2 protein. Based on the model, residues Ser299, Asp107, and Glu109 interact with the substrate and potential epitopes (five conformational and seven linear) located at surface-exposed regions of the structure. Purified native Pp-Hyal showed high similarity (97%) with hyaluronidase from Polistes annularis venom (Q9U6V9). Immunoblotting analysis confirmed the specificity of the Pp-Hyal-specific antibody as it recognized the Pp-Hyal protein in both the purified fraction and P. paulista crude venom. No reaction was observed with the venoms of Apis mellifera, Solenopsis invicta, Agelaia pallipes pallipes, and Polistes lanio lanio, with the exception of immune cross-reactivity with venoms of the genus Polybia (sericea and ignobilis). Our results demonstrate cross-reactivity only between wasp venoms from the genus Polybia. The absence of cross-reactivity between the venoms of wasps and bees observed here is important because it allows identification of the insect responsible for sensitization, or at least of the phylogenetically closest insect, in order to facilitate effective immunotherapy in allergic patients. © 2013 Elsevier Ltd.

Adsorption of polyelectrolytes onto oppositely charged cylindrical macroions

In this work we investigate the adsorption of polyelectrolyte chains onto uniformly charged cylindrical macroions by means of the Metropolis Monte Carlo simulations and weighted histogram analysis method. Adopting a simplified model for macromolecules and treating the electrolytic solution in the Debye-Hückel level, conformational properties of the adsorbed chain, such as the radius of gyration and the thickness of the adsorbed layer, are provided as a function of ionic strength and macroion charge density. By analysis of the free energy profile as a function of the radius of gyration it was possible to identify first-order-like transitions between adsorbed and desorbed states and obtain a macroion charge density dependence of the critical ionic strength in good agreement with experiments. © 2013 AIP Publishing LLC.

Elucidation of carbohydrate molecular interaction mechanism of recombinant and native ArtinM

The quartz crystal microbalance (QCM) technique has been applied for monitoring the biorecognition of ArtinM lectins at low horseradish peroxidase glycoprotein (HRP) concentrations, using a simple kinetic model based on Langmuir isotherm in previous work.18 The latter approach was consistent with the data at dilute conditions but it fails to explain the small differences existing in the jArtinM and rArtinM due to ligand binding concentration limit. Here we extend this analysis to differentiate sugar-binding event of recombinant (rArtinM) and native (jArtinM) ArtinM lectins beyond dilute conditions. Equivalently, functionalized quartz crystal microbalance with dissipation monitoring (QCM-D) was used as real-time label-free technique but structural-dependent kinetic features of the interaction were detailed by using combined analysis of mass and dissipation factor variation. The stated kinetic model not only was able to predict the diluted conditions but also allowed to differentiate ArtinM avidities. For instance, it was found that rArtinM avidity is higher than jArtinM avidity whereas their conformational flexibility is lower. Additionally, it was possible to monitor the hydration shell of the binding complex with ArtinM lectins under dynamic conditions. Such information is key in understanding and differentiating protein binding avidity, biological functionality, and kinetics. © 2013 American Chemical Society.

Development and Biotechnological Application of a Novel Endoxylanase Family GH10 Identified from Sugarcane Soil Metagenome

Metagenomics has been widely employed for discovery of new enzymes and pathways to conversion of lignocellulosic biomass to fuels and chemicals. In this context, the present study reports the isolation, recombinant expression, biochemical and structural characterization of a novel endoxylanase family GH10 (SCXyl) identified from sugarcane soil metagenome. The recombinant SCXyl was highly active against xylan from beechwood and showed optimal enzyme activity at pH 6,0 and 45°C. The crystal structure was solved at 2.75 Å resolution, revealing the classical (β/α)8-barrel fold with a conserved active-site pocket and an inherent flexibility of the Trp281-Arg291 loop that can adopt distinct conformational states depending on substrate binding. The capillary electrophoresis analysis of degradation products evidenced that the enzyme displays unusual capacity to degrade small xylooligosaccharides, such as xylotriose, which is consistent to the hydrophobic contacts at the +1 subsite and low-binding energies of subsites that are distant from the site of hydrolysis. The main reaction products from xylan polymers and phosphoric acid-pretreated sugarcane bagasse (PASB) were xylooligosaccharides, but, after a longer incubation time, xylobiose and xylose were also formed. Moreover, the use of SCXyl as pre-treatment step of PASB, prior to the addition of commercial cellulolytic cocktail, significantly enhanced the saccharification process. All these characteristics demonstrate the advantageous application of this enzyme in several biotechnological processes in food and feed industry and also in the enzymatic pretreatment of biomass for feedstock and ethanol production. © 2013 Alvarez et al.

Analyzing the effect of homogeneous frustration in protein folding

The energy landscape theory has been an invaluable theoretical framework in the understanding of biological processes such as protein folding, oligomerization, and functional transitions. According to the theory, the energy landscape of protein folding is funneled toward the native state, a conformational state that is consistent with the principle of minimal frustration. It has been accepted that real proteins are selected through natural evolution, satisfying the minimum frustration criterion. However, there is evidence that a low degree of frustration accelerates folding. We examined the interplay between topological and energetic protein frustration. We employed a Cα structure-based model for simulations with a controlled nonspecific energetic frustration added to the potential energy function. Thermodynamics and kinetics of a group of 19 proteins are completely characterized as a function of increasing level of energetic frustration. We observed two well-separated groups of proteins: one group where a little frustration enhances folding rates to an optimal value and another where any energetic frustration slows down folding. Protein energetic frustration regimes and their mechanisms are explained by the role of non-native contact interactions in different folding scenarios. These findings strongly correlate with the protein free-energy folding barrier and the absolute contact order parameters. These computational results are corroborated by principal component analysis and partial least square techniques. One simple theoretical model is proposed as a useful tool for experimentalists to predict the limits of improvements in real proteins. © 2013 Wiley Periodicals, Inc.