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Protein Surface (protein + surface)
Selected AbstractsA novel thermostable hemoglobin from the actinobacterium Thermobifida fuscaFEBS JOURNAL, Issue 16 2005Alessandra Bonamore The gene coding for a hemoglobin-like protein (Tf-trHb) has been identified in the thermophilic actinobacterium Thermobifida fusca and cloned in Escherichia coli for overexpression. The crystal structure of the ferric, acetate-bound derivative, was obtained at 2.48 Å resolution. The three-dimensional structure of Tf-trHb is similar to structures reported for the truncated hemoglobins from Mycobacterium tuberculosis and Bacillus subtilis in its central domain. The complete lack of diffraction patterns relative to the N- and C-terminal segments indicates that these are unstructured polypeptides chains, consistent with their facile cleavage in solution. The absence of internal cavities and the presence of two water molecules between the bound acetate ion and the protein surface suggest that the mode of ligand entry is similar to that of typical hemoglobins. The protein is characterized by higher thermostability than the similar mesophilic truncated hemoglobin from B. subtilis, as demonstrated by far-UV CD melting experiments on the cyano-met derivatives. The ligand-binding properties of Tf-trHb, analyzed in stopped flow experiments, demonstrate that Tf-trHb is capable of efficient O2 binding and release between 55 and 60 °C, the optimal growth temperature for Thermobifida fusca. [source] Mutations towards enantioselectivity adversely affect secretion of Pseudomonas aeruginosa lipaseFEMS MICROBIOLOGY LETTERS, Issue 1 2008Sascha Hausmann Abstract Lipases are important biocatalysts used as detergent additives to manufacture biodiesel, and in particular, for the production of enantiopure compounds such as alcohols, amines and carboxylic acids. Extensive efforts were conducted trying to optimize lipase properties and lipase LipA of Pseudomonas aeruginosa comprises the best-studied example in terms of optimizing enantioselectivity by application of numerous directed evolution methods. Its enantioselectivity in the asymmetric hydrolysis of the model substrate 2-methyldecanoic acid p -nitrophenyl ester was increased from E=1.1 for the wild-type enzyme to E=51 for the best (S)-enantioselective variant which carried six amino acid exchanges. We have observed that overexpression of this variant in the homologous host resulted in only marginal yields of enzyme in the bacterial culture supernatant, suggesting that the enantioselective LipA variant was secreted with only low efficiency. Hence, we have analysed the secretion of this lipase variant and compared it to variants carrying either the respective single mutations or some combinations. We report here the identification of two amino acid substitutions located on the protein surface, which significantly impair lipase secretion. [source] Influence of Decreasing Solvent Polarity (1,4-Dioxane/Water Mixtures) on the Acid,Base and Copper(II)-Binding Properties of Guanosine 5,-Diphosphate,HELVETICA CHIMICA ACTA, Issue 3 2005Emanuela The acidity constants of twofold protonated guanosine 5,-diphosphate, H2(GDP),, and the stability constants of the [Cu(H;GDP)] and [Cu(GDP)], complexes were determined in H2O as well as in 30 or 50% (v/v) 1,4-dioxane/H2O by potentiometric pH titrations (25°; I=0.1M, NaNO3). The results showed that in H2O one of the two protons of H2(GDP), is located mainly at the N(7) site and the other one at the terminal , -phosphate group. In contrast, for 50% 1,4-dioxane/H2O solutions, a micro acidity-constant evaluation evidenced that ca. 75% of the H2(GDP), species have both protons phosphate-bound, because the basicity of pyridine-type N sites decreases with decreasing solvent polarity whereas the one of phosphate groups increases. In the [Cu(H;GDP)] complex, the proton and the metal ion are in all three solvents overwhelmingly phosphate-bound, and the release of this proton is inhibited by decreasing polarity of the solvent. Based on previously determined straight-line plots of log,Kvs. pK (where R represents a non-interacting residue in simple diphosphate monoesters ROP(O,)(O)OP(O)(O,)2, RDP3,), which were now extended to mixed solvents (based on analogies), it is concluded that, in all three solvents, the [Cu(GDP)], complex is more stable than expected based on the basicity of the diphosphate residue. This increased stability is attributed to macrochelate formation of the phosphate-coordinated Cu2+ with N(7) of the guanine residue. The formation degree of this macrochelate amounts in aqueous solution to ca. 75% (being thus higher than that of the Cu2+ complex of adenosine 5,-diphosphate) and in 50% (v/v) 1,4-dioxane/H2O to ca. 60%, i.e., the formation degree of the macrochelate is only relatively little affected by the change in solvent, though it needs to be emphasized that the overall stability of the [Cu(GDP)], complex increases with decreasing solvent polarity. By including previously studied systems in the considerations, the biological implications are shortly discussed, and it is concluded that Nature has here a tool to alter the structure of complexes by shifting them on a protein surface from a polar to an apolar region and vice versa. [source] Characterisation of FXTAS related isolated intranuclear protein inclusions using laser tweezers Raman spectroscopyJOURNAL OF RAMAN SPECTROSCOPY, Issue 1 2010Tobias J. Moritz Abstract We report the analysis of the vibrational modes of intranuclear protein inclusions isolated from the brain of human subjects with the Fragile X-associated tremor/ataxia syndrome (FXTAS). In this preliminary study, Raman spectra of optically trapped inclusions were measured and analysed to determine protein composition and structure. Our main findings are as follows: (1) The spectra of protein inclusions are characteristic of H2A and H2B histones, which correlate with previous mass spectrometry (MS) studies; (2) Tyrosine is present in its OH form and exposed at the protein surface; (3) Zn and to a lesser extent Cu bound to histidine side chains are detected in the inclusions; (4) The tryptophan side-chain torsion angle is calculated to be 102°; (5) Several potential spectroscopic markers for the inclusions of FXTAS are identified. These results show the capability of using laser tweezers Raman spectroscopy to identify protein inclusions in a non-perturbative way and to gain further insight into the pathogenesis and progression of FXTAS in human subjects and in experimental models of this disorder. Copyright © 2009 John Wiley & Sons, Ltd. [source] Activity and regulation of glycoPEGylated factor VIIa analogsJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 9 2008S. GHOSH Summary.,Background:,Recombinant coagulation factor VIIa (rFVIIa) has proven to be a safe and effective drug for treatment of bleeding episodes in hemophilic patients with inhibitors. However, rFVIIa is cleared from the circulation relatively quickly. Protein modification with poly(ethylene glycol) (PEG) can prolong the circulatory lifetime of proteins but it could also impair protein function by molecular shielding of the protein surface. Objectives:, To characterize the interaction of glycoPEGylated rFVIIa , rFVIIa-10K PEG and rFVIIa-40K PEG , with tissue factor (TF), factor X (FX) and plasma inhibitors, tissue factor pathway inhibitor (TFPI) and antithrombin (AT). Methods:, The amidolytic and FX activation assays were employed to investigate the interaction of glycoPEGylated rFVIIa with its macromolecular substrate and inhibitors. Results:, Both the glycoPEGylated rFVIIa analogs exhibited similar amidolytic activity as that of rFVIIa in the absence or the presence of relipidated TF. The analogs were as effective as rFVIIa in activating FX in the absence of TF. In the presence of TF, the glycoPEGylated rFVIIa variants, relative to rFVIIa, were slightly less effective at lower concentrations, but no significant differences were found among them in activating FX at saturating concentrations. Both AT/heparin and TFPI effectively inhibited the glycoPEGylated rFVIIa bound to relipidated TF or TF on stimulated endothelial cells. In contrast to their normal interaction with TF, the glycoPEGylated rFVIIa variants appeared to interact poorly with phospholipids. Conclusions:, The glycoPEGylated rFVIIa variants retained their catalytic activity and interacted efficiently with TF, FX and the plasma inhibitors. Further work with appropriate in vitro and in vivo model systems is needed to determine the feasibility of using glycoPEGylated rFVIIa to improve therapeutic options for bleeding disorders. [source] Nature versus nurture in two highly enantioselective esterases from Bacillus cereus and Thermoanaerobacter tengcongensisMICROBIAL BIOTECHNOLOGY, Issue 1 2010Stephan Grosse Summary There is an increasing need for the use of biocatalysis to obtain enantiopure compounds as chiral building blocks for drug synthesis such as antibiotics. The principal findings of this study are: (i) the complete sequenced genomes of Bacillus cereus ATCC 14579 and Thermoanaerobacter tengcongensis MB4 contain a hitherto undescribed enantioselective and alkaliphilic esterase (BcEST and TtEST respectively) that is specific for the production of (R)-2-benzyloxy-propionic acid ethyl ester, a key intermediate in the synthesis of levofloxacin, a potent antibiotic; and (ii) directed evolution targeted for increased thermostability of BcEST produced two improved variants, but in either case the 3,5°C increase in the apparent melting temperature (Tm) of the mutants over the native BcEST that has a Tm of 50°C was outperformed by TtEST, a naturally occurring homologue with a Tm of 65°C. Protein modelling of BcEST mapped the S148C and K272R mutations at protein surface and the I88T and Q110L mutations at more buried locations. This work expands the repertoire of characterized members of the ,/,-fold hydrolase superfamily. Further, it shows that genome mining is an economical option for new biocatalyst discovery and we provide a rare example of a naturally occurring thermostable biocatalyst that outperforms experimentally evolved homologues that carry out the same hydrolysis. [source] Structural and dynamic properties of water around acetylcholinesterasePROTEIN SCIENCE, Issue 9 2002Richard H. Henchman AChE, acetylcholinsterase; ARC/TAP, averaged residue coordinate/time-averaged position Abstract Structural and dynamic properties of water molecules around acetylcholinesterase are examined from a 10-nsec molecular dynamics simulation to help understand how the protein alters water properties. Water structure is broken down into hydration sites constructed from the water density <3.6 Å from the protein surface. These sites are characterized according to occupancy, number of water neighbors, hydrogen bonds, dipole moment, and residence time. The site description provides a convenient means to describe the extent and localization of these properties. Determining the network of paths that waters follow from site to site and measuring the rate of flow of waters from the sites to the bulk make it possible to quantitatively study the time scales and paths that water molecules follow as they move around the protein. [source] Structure and dynamics of translation initiation factor aIF-1A from the archaeon Methanococcus jannaschii determined by NMR spectroscopyPROTEIN SCIENCE, Issue 12 2001Wei Li Abstract Translation initiation factor 1A (aIF-1A) from the archaeon Methanococcus jannaschii was expressed in Escherichia coli, purified, and characterized in terms of its structure and dynamics using multidimensional NMR methods. The protein was found to be a member of the OB-fold family of RNA-associated proteins, containing a barrel of five beta-strands, a feature that is shared with the homologous eukaryotic translation initiation factor 1A (eIF-1A), as well as the prokaryotic translation initiation factor IF1. External to the , barrel, aIF-1A contains an ,-helix at its C-terminal and a flexible loop at its N-terminal, features that are qualitatively similar to those found in eIF-1A, but not present in prokaryotic IF1. The structural model of aIF-1A, when used in combination with primary sequence information for aIF-1A in divergent species, permitted the most-conserved residues on the protein surface to be identified, including the most likely candidates for direct interaction with the 16S ribosomal RNA and other components of the translational apparatus. Several of the conserved surface residues appear to be unique to the archaea. Nitrogen-15 relaxation and amide exchange rate data were used to characterize the internal motions within aIF-1A, providing evidence that the protein surfaces that are most likely to participate in intermolecular interactions are relatively flexible. A model is proposed, suggesting some specific interactions that may occur between aIF-1A and the small subunit of the archaeal ribosome. [source] Ligand diffusion in the catalase from Proteus mirabilis: A molecular dynamics studyPROTEIN SCIENCE, Issue 10 2001Patricia Amara Abstract The role of the channels and cavities present in the catalase from Proteus mirabilis (PMC) was investigated using molecular dynamics (MD) simulations. The reactant and products of the reaction, H2O2 ,1/2 O2 + H2O, catalyzed by the enzyme were allowed to diffuse to and from the active site. Dynamic fluctuations in the structure are found necessary for the opening of the major channel, ideied in the X-ray model, which allows access to the active site. This channel is the only pathway to the active site observed during the dynamics, and both the products and reactant use it. H2O and O2 are also detected in a cavity defined by the heme and Ser196, which could play an important role during the reaction. Free energy profiles of the ligands diffusing through the major channel indicate that the barriers to ligand diffusion are less than 20 kJ mol,1 for each of the species. It is not clear from our study that minor channels play a role for access to the protein active site or to the protein surface. [source] Atomic resolution studies of carbonic anhydrase IIACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2010Craig A. Behnke Carbonic anhydrase has been well studied structurally and functionally owing to its importance in respiration. A large number of X-ray crystallographic structures of carbonic anhydrase and its inhibitor complexes have been determined, some at atomic resolution. Structure determination of a sulfonamide-containing inhibitor complex has been carried out and the structure was refined at 0.9,Å resolution with anisotropic atomic displacement parameters to an R value of 0.141. The structure is similar to those of other carbonic anhydrase complexes, with the inhibitor providing a fourth nonprotein ligand to the active-site zinc. Comparison of this structure with 13 other atomic resolution (higher than 1.25,Å) isomorphous carbonic anhydrase structures provides a view of the structural similarity and variability in a series of crystal structures. At the center of the protein the structures superpose very well. The metal complexes superpose (with only two exceptions) with standard deviations of 0.01,Å in some zinc,protein and zinc,ligand bond lengths. In contrast, regions of structural variability are found on the protein surface, possibly owing to flexibility and disorder in the individual structures, differences in the chemical and crystalline environments or the different approaches used by different investigators to model weak or complicated electron-density maps. These findings suggest that care must be taken in interpreting structural details on protein surfaces on the basis of individual X-ray structures, even if atomic resolution data are available. [source] Structures of the multicomponent Rieske non-heme iron toluene 2,3-dioxygenase enzyme systemACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2009Rosmarie Friemann Bacterial Rieske non-heme iron oxygenases catalyze the initial hydroxylation of aromatic hydrocarbon substrates. The structures of all three components of one such system, the toluene 2,3-dioxygenase system, have now been determined. This system consists of a reductase, a ferredoxin and a terminal dioxygenase. The dioxygenase, which was cocrystallized with toluene, is a heterohexamer containing a catalytic and a structural subunit. The catalytic subunit contains a Rieske [2Fe,2S] cluster and mononuclear iron at the active site. This iron is not strongly bound and is easily removed during enzyme purification. The structures of the enzyme with and without mononuclear iron demonstrate that part of the structure is flexible in the absence of iron. The orientation of the toluene substrate in the active site is consistent with the regiospecificity of oxygen incorporation seen in the product formed. The ferredoxin is Rieske type and contains a [2Fe,2S] cluster close to the protein surface. The reductase belongs to the glutathione reductase family of flavoenzymes and consists of three domains: an FAD-binding domain, an NADH-binding domain and a C-terminal domain. A model for electron transfer from NADH via FAD in the reductase and the ferredoxin to the terminal active-site mononuclear iron of the dioxygenase is proposed. [source] MAD phasing using the (Ta6Br12)2+ cluster: a retrospective studyACTA CRYSTALLOGRAPHICA SECTION D, Issue 5 2008Oliwia Pasternak The crystal structure of cytokinin-specific binding protein (CSBP) containing four independent molecules with 4 × 155 = 620 residues in the asymmetric unit of the P64 unit cell has been solved by three-wavelength MAD using 1.8,Å resolution data recorded from a crystal derivatized with the dodecabromohexatantalum cation (Ta6Br12)2+. The diffraction data contained a very strong anomalous signal (allowing successful phasing even using peak SAD data alone) despite the fact that the five (Ta6Br12)2+ clusters found in the asymmetric unit have low occupancy (about 0.3). The derivative structure has been successfully refined to R = 0.158, providing interesting details on the geometry of the (Ta6Br12)2+ cluster, its interactions with the protein and on the backsoaking of a cytokinin ligand that was originally part of a CSBP,cytokinin complex in the native crystals used for (Ta6Br12)2+ derivatization. A simulation analysis of the phasing power of the (Ta6Br12)2+ ions at artificially imposed resolution limits shows that it is not possible to resolve the individual Ta atoms if the dmin limit of the data is higher than 2.9,Å. Additionally, for successful Ta identification the (Ta6Br12)2+ complex should be specifically bound and ordered. Good binding at the protein surface is facilitated by the presence of acidic groups, indicating higher pH buffer conditions to be preferable. In addition, the water channels in the crystal should be sufficiently wide (at least 11,Å) to allow free diffusion of the (Ta6Br12)2+ ions on soaking. A retrospective look at the initial molecular-replacement calculations provides interesting insights into how the peculiar packing mode and strong bias of the molecular-replacement-phased electron-density maps had hindered successful solution of the structure by this method. [source] Macromolecular recognition in the Protein Data BankACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2007Joël Janin Crystal structures deposited in the Protein Data Bank illustrate the diversity of biological macromolecular recognition: transient interactions in protein,protein and protein,DNA complexes and permanent assemblies in homodimeric proteins. The geometric and physical chemical properties of the macromolecular interfaces that may govern the stability and specificity of recognition are explored in complexes and homodimers compared with crystal-packing interactions. It is found that crystal-packing interfaces are usually much smaller; they bury fewer atoms and are less tightly packed than in specific assemblies. Standard-size interfaces burying 1200,2000,Å2 of protein surface occur in protease,inhibitor and antigen,antibody complexes that assemble with little or no conformation changes. Short-lived electron-transfer complexes have small interfaces; the larger size of the interfaces observed in complexes involved in signal transduction and homodimers correlates with the presence of conformation changes, often implicated in biological function. Results of the CAPRI (critical assessment of predicted interactions) blind prediction experiment show that docking algorithms efficiently and accurately predict the mode of assembly of proteins that do not change conformation when they associate. They perform less well in the presence of large conformation changes and the experiment stimulates the development of novel procedures that can handle such changes. [source] Crystal quality and differential crystal-growth behaviour of three proteins crystallized in gel at high hydrostatic pressureACTA CRYSTALLOGRAPHICA SECTION D, Issue 6 2005A. Kadri Pressure is a non-invasive physical parameter that can be used to control and influence protein crystallization. It is also found that protein crystals of superior quality can be produced in gel. Here, a novel crystallization strategy combining hydrostatic pressure and agarose gel is described. Comparative experiments were conducted on hen and turkey egg-white lysozymes and the plant protein thaumatin. Crystals could be produced under up to 75,100,MPa (lysozymes) and 250,MPa (thaumatin). Several pressure-dependent parameters were determined, which included solubility and supersaturation of the proteins, number, size and morphology of the crystals, and the crystallization volume. Exploration of three-dimensional phase diagrams in which pH and pressure varied identified growth conditions where crystals had largest size and best morphology. As a general trend, nucleation and crystal-growth kinetics are altered and nucleation is always enhanced under pressure. Further, solubility of the lysozymes increases with pressure while that of thaumatin decreases. Likewise, changes in crystallization volumes at high and atmospheric pressure are opposite, being positive for the lysozymes and negative for thaumatin. Crystal quality was estimated by analysis of Bragg reflection profiles and X-ray topographs. While the quality of lysozyme crystals deteriorates as pressure increases, that of thaumatin crystals improves, with more homogeneous crystal morphology suggesting that pressure selectively dissociates ill-formed nuclei. Analysis of the thaumatin structure reveals a less hydrated solvent shell around the protein when pressure increases, with ,20% less ordered water molecules in crystals grown at 150,MPa when compared with those grown at atmospheric pressure (0.1,MPa). Noticeably, the altered water distribution is seen in depressurized crystals, indicating that pressure triggers a stable structural alteration on the protein surface while its polypeptide backbone remains essentially unaltered. [source] Asymmetries in the nucleosome core particle at 2.5,Å resolutionACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2000Joel M. Harp The 2.5,Å X-ray crystal structure of the nucleosome core particle presented here provides significant additions to the understanding of the nucleosome, the fundamental unit of chromatin structure. Extensions are made to the structure of the N-terminal histone tails and details are provided on hydration and ion binding. The structure is composed of twofold symmetric molecules, native chicken histone octamer cores and the DNA palindrome, which were expected to form a perfectly twofold symmetric nucleosome core particle. In fact, the result is asymmetric owing to the binding of the DNA to the protein surface and to the packing of the particles in the crystal lattice. An analysis is made of the asymmetries by comparisons both within the nucleosome core particle and to the structure of the histone octamer core of the nucleosome. [source] Revisiting glutaraldehyde cross-linking: the case of the Arg,Lys intermolecular doubletACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2010Michèle Salem In addition to the common use of glutaraldehyde to nonspecifically cross-link protein crystals through lysine residues disposed on the surface of the protein, the use of gentle vapour diffusion of glutaraldehyde offers a convenient way to limit polymerization and to allow slow diffusion throughout the crystal. In the case of trimeric barnase crystals, a specific cross-link was observed between an lysine side chain and an arginine side chain that were spatially disposed at the ideal distance on the protein surface in the three monomers. Here, the direct observation of a specific Lys,Arg cross-link site is reported and a mechanism is proposed for the reaction. [source] Truncated hemoglobins: trimming the classical ,three-over-three' globin fold to a minimal sizeBIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 3 2001Mario Milani Abstract Truncated hemoglobins (trHbs) host the heme in a ,two-over-two' ,-helical sandwich which results from extensive editing of the classical ,three-over-three' globin fold. The three-dimensional structure of trHbs is based on four main ,-helices, arranged in a sort of ,-helical bundle composed of two antiparallel helix pairs (B/E and G/H). Most notably, trHbs deviate from the conventional globin fold in that they display an extended loop substituting for the heme proximal F-helix observed in globins. Moreover, since efficient adaptation of a 110,130 amino acid trHb chain to host the porphyrin ring firstly requires specific chain flexibility, trHbs contain three invariant Gly-based motifs. Inspection of the trHb three-dimensional trHb structures shows that an apparent protein cavity or tunnel would connect the protein surface to an inner region very close to the heme distal site. Such a structural feature, never observed before in (non) vertebrate globins, may have substantial implications for ligand diffusion and binding properties in trHbs. © 2001 IUBMB. Published by Elsevier Science Ltd. All rights reserved. [source] Investigation of protein binding affinity and preferred orientations in ion exchange systems using a homologous protein libraryBIOTECHNOLOGY & BIOENGINEERING, Issue 3 2009Wai Keen Chung Abstract A library of cold shock protein B (CspB) mutant variants was employed to study protein binding affinity and preferred orientations in cation exchange chromatography. Single site mutations introduced at charged amino acids on the protein surface resulted in a homologous protein set with varying charge density and distribution. The retention times of the mutants varied significantly during linear gradient chromatography. While the expected trends were observed with increasing or decreasing positive charge on the protein surface, the degree of change was a strong function of the location and microenvironment of the mutated amino acid. Quantitative structure,property relationship (QSPR) models were generated using a support vector regression technique that was able to give good predictions of the retention times of the various mutants. Molecular descriptors selected during model generation were used to elucidate the factors affecting protein retention. Electrostatic potential maps were also employed to provide insight into the effects of protein surface topography, charge density and charge distribution on protein binding affinity and possible preferred binding orientations. The use of this protein mutant library in concert with the qualitative and quantitative analyses presented in the article provides an improved understanding of protein behavior in ion exchange systems. Biotechnol. Bioeng. 2009; 102: 869,881. © 2008 Wiley Periodicals, Inc. [source] Studies of lysozyme binding to histamine as a ligand for hydrophobic charge induction chromatographyBIOTECHNOLOGY PROGRESS, Issue 1 2010Qing-Hong Shi Abstract Histamine was immobilized on Sepharose CL-6B (Sepharose) for use as a ligand of hydrophobic charge induction chromatography (HCIC) of proteins. Lysozyme adsorption onto Histamine-Sepharose (HA-S) was studied by adsorption equilibrium and calorimetry to uncover the thermodynamic mechanism of the protein binding. In both the experiments, the influence of salt (ammonium sulfate and sodium sulfate) was examined. Adsorption isotherms showed that HA-S exhibited a high salt tolerance in lysozyme adsorption. This property was well explained by the combined contributions of hydrophobic interaction and aromatic stacking. The isotherms were well fitted to the Langmuir equation, and the equilibrium parameters for lysozyme adsorption were obtained. In addition, thermodynamic parameters (,Hads, ,Sads, and ,Gads) for the adsorption were obtained by isothermal titration calorimetry by titrating lysozyme solutions into the adsorbent suspension. Furthermore, free histamine was titrated into lysozyme solution in the same salt-buffers. Compared with the binding of lysozyme to free histamine, lysozyme adsorption onto HA-S was characterized by a less favorable ,Gads and an unfavorable ,Sads because histamine was covalently attached to Sepharose via a three-carbon-chain spacer. Consequently, the immobilized histamine could only associate with the residues on the protein surface rather than those in the hydrophobic pocket, causing a less favorable orientation between histamine and lysozyme. Further comparison of thermodynamic parameters indicated that the unfavorable ,Sads was offset by a favorable ,Hads, thus exhibiting typical enthalpy-entropy compensation. Moreover, thermodynamic analyses indicated the importance of the dehydration of lysozyme molecule and HA-S during the adsorption and a substantial conformational change of the protein during adsorption. The results have provided clear insights into the adsorption mechanisms of lysozyme onto the new HCIC material. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010 [source] Osmolyte controlled fibrillation kinetics of insulin: New insight into fibrillation using the preferential exclusion principleBIOTECHNOLOGY PROGRESS, Issue 5 2009Arpan Nayak Abstract Amyloid proteins are converted from their native-fold to long ,-sheet-rich fibrils in a typical sigmoidal time-dependent protein aggregation curve. This reaction process from monomer or dimer to oligomer to nuclei and then to fibrils is the subject of intense study. The main results of this work are based on the use of a well-studied model amyloid protein, insulin, which has been used in vitro by others. Nine osmolyte molecules, added during the protein aggregation process for the production of amyloid fibrils, slow-down or speed up the process depending on the molecular structure of each osmolyte. Of these, all stabilizing osmolytes (sugars) slow down the aggregation process in the following order: tri > di > monosaccharides, whereas destabilizing osmolytes (urea, guanidium hydrochloride) speed up the aggregation process in a predictable way that fits the trend of all osmolytes. With respect to kinetics, we illustrate, by adapting our earlier reaction model to the insulin system, that the intermediates (trimers, tetramers, pentamers, etc.) are at very low concentrations and that nucleation is orders of magnitude slower than fibril growth. The results are then collated into a cogent explanation using the preferential exclusion and accumulation of osmolytes away from and at the protein surface during nucleation, respectively. Both the heat of solution and the neutral molecular surface area of the osmolytes correlate linearly with two fitting parameters of the kinetic rate model, that is, the lag time and the nucleation rate prior to fibril formation. These kinetic and thermodynamic results support the preferential exclusion model and the existence of oligomers including nuclei and larger structures that could induce toxicity. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009 [source] Thiopropyl-agarose as a solid phase reducing agent for chemical modification of IgG and F(ab,)2BIOTECHNOLOGY PROGRESS, Issue 5 2008Natalia Ferraz Abstract Selective reduction of native disulfide bonds in immunoglobulins is one of the best methods for introducing reactive groups on to the protein surface. Additionally, the thiol groups so generated may allow oriented conjugation at a specific site of the immunoglobulin. Solid-phase reducing agents have many advantages over soluble ones (including ease of separation of excess reagent from reduced protein by filtration, and the potential for regeneration and multiple reuse). In this work we report a comparative study of the reduction of rabbit IgG and its F(ab,)2 fragments, with mercaptohydroxypropylether-agarose (thiopropyl-agarose), a solid phase reducing agent, and dithiothreitol. The effect of different parameters on the process, such as the amount of reducing agent, incubation period, and temperature, was assessed by titration of thiol groups and SDS-PAGE analysis. Optimized reduction with thiopropyl-agarose introduced six thiol groups in the F(ab,)2 fragment (mol/mol). Native IgG was less reactive, probably due to steric effects, as only an average of three thiol groups were introduced. However, by increasing reaction temperature from 22 to 37°C, six thiol groups could be introduced in native IgG (mol/mol). Reduction with dithiothreitol also introduced six thiol groups in F(ab,)2 fragments (mol/mol) but led to higher thiol content for the whole IgG. These results demonstrated that thiopropyl-agarose can be a very useful tool for exercising more precise control over the reduction treatment, and for selecting which disulfide bridges are to be broken. After 6 h incubation with reducing agent containing 8 and 16 ,moles SH per mg of protein, the resulting reduced IgG retained the same biological activity as the native immunoglobulin. The controlled modification of native disulfides achieved with thiopropyl-agarose will be useful for the development of soluble and insoluble immunoglobulin conjugates. [source] Molecular Mechanism of the Hydration of Candida antarctica Lipase B in the Gas Phase: Water Adsorption Isotherms and Molecular Dynamics SimulationsCHEMBIOCHEM, Issue 18 2009Ricardo J. F. Branco Dr. Abstract Hydration is a major determinant of activity and selectivity of enzymes in organic solvents or in gas phase. The molecular mechanism of the hydration of Candida antarctica lipase B (CALB) and its dependence on the thermodynamic activity of water (aw) was studied by molecular dynamics simulations and compared to experimentally determined water sorption isotherms. Hydration occurred in two phases. At low water activity, single water molecules bound to specific water binding sites at the protein surface. As the water activity increased, water networks gradually developed. The number of protein-bound water molecules increased linearly with aw, until at aw=0.5 a spanning water network was formed consisting of 311 water molecules, which covered the hydrophilic surface of CALB, with the exception of the hydrophobic substrate-binding site. At higher water activity, the thickness of the hydration shell increased up to 10 Å close to aw=1. Above a limit of 1600 protein-bound water molecules the hydration shell becomes unstable and the formation of pure water droplets occurs in these oversaturated simulation conditions. While the structure and the overall flexibility of CALB was independent of the hydration state, the flexibility of individual loops was sensitive to hydration: some loops, such as those part of the substrate-binding site, became more flexible, while other parts of the protein became more rigid upon hydration. However, the molecular mechanism of how flexibility is related to activity and selectivity is still elusive. [source] Early Structural Evolution of Native Cytochrome c after Solvent RemovalCHEMBIOCHEM, Issue 15 2008Michal Z. Steinberg Abstract Electrospray ionization transfers thermally labile biomolecules, such as proteins, from solution into the gas phase, where they can be studied by mass spectrometry. Covalent bonds are generally preserved during and after the phase transition, but it is less clear to what extent noncovalent interactions are affected by the new gaseous environment. Here, we present atomic-level computational data on the structural rearrangement of native cytochrome c immediately after solvent removal. The first structural changes after desolvation occur surprisingly early, on a timescale of picoseconds. For the time segment of up to 4.2 ns investigated here, we observed no significant breaking of native noncovalent bonds; instead, we found formation of new noncovalent bonds. This generally involves charged residues on the protein surface, resulting in transiently stabilized intermediate structures with a global fold that is essentially the same as that in solution. Comparison with data from native electron capture dissociation experiments corroborates both its mechanistic postulations and our computational predictions, and suggests that global structural changes take place on a millisecond timescale not covered by our simulations. [source] Exo-Mechanism Proximity-Accelerated Alkylations: Investigations of Linkers, Electrophiles and Surface Mutations in Engineered Cyclophilin,Cyclosporin SystemsCHEMBIOCHEM, Issue 5 2005Konstantin Levitsky Abstract Investigations on the scope and utility of exo-mechanism proximity-accelerated reactions in engineered receptor,ligand systems are reported. We synthesized a series of electrophilic cyclosporin (CsA) derivatives by varying electrophiles and linker lengths, prepared a series of nucleophilic cysteine mutations on the surface of cyclophilin A (Cyp), and examined their reactivity and specificity in proximity-accelerated reactions. Acrylamide and epoxide electrophiles afforded useful reactivity and high specificity for alkylation of engineered receptors in Jurkat cell extracts. We found that remote cysteines (>17 Å from the ligand) could be alkylated with useful rates under physiological conditions. The results from mutations of the receptor surface suggest that the dominant factors governing the rates of proximity-accelerated reactions are related to the local environment of the reactive group on the protein surface. This study defines several parameters affecting reactivity in exo-mechanism proximity-accelerated reactions and provides guidance for the design of experiments for biological investigations involving proximity-accelerated reactions. [source] Regulation of ,-Chymotrypsin Catalysis by Ferric Porphyrins and CyclodextrinsCHEMISTRY - AN ASIAN JOURNAL, Issue 4 2008Koji Kano Prof. Abstract Positively charged ,-chymotrypsin (ChT) formed a 1:1 complex with negatively charged 5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato iron(III) (FeTPPS) in phosphate buffer at pH,7.4 through electrostatic interaction. In spite of the large binding constant (K=4.8×105,M,1), FeTPPS could not completely inhibit the catalysis of ChT in the hydrolysis of the model substrate, N -succinyl- L -phenylalanine p -nitroanilide (SPNA). The degree of inhibition (60,%) was saturated at 1.6,equivalents of FeTPPS, which indicates that covering of the active site of ChT by FeTPPS was insufficient. The enzymatic activity lowered by FeTPPS was entirely recovered for the freshly prepared sample when the porphyrin on the protein surface was detached by per- O -methylated ,-cyclodextrin (TMe-,-CD), which formed a stable 1:2 inclusion complex with FeTPPS (K1=1.26×106,M,1, K2=6.3×104,M,1). FeTPPS gradually induced irreversible denaturation of ChT, and the denatured ChT further lost its catalytic ability. No repairing effect of TMe-,-CD was observed with irreversibly denatured ChT. A new reversible inhibitor, 5,10,15,20-tetrakis[4-(3,5-dicarboxyphenylmethoxy)phenyl]porphyrinato iron(III) (FeP8M), was then designed, and its inhibitory behavior was examined. FeP8M formed very stable 1:1 and 1:2 FeP8M/ChT complexes with ChT, the K1 and K2 values being 2.0×108 and 1.0×106,M,1, respectively. FeP8M effectively inhibited the ChT-catalyzed hydrolysis of SPNA (maximum degree of inhibition=85,%), and the activity of ChT was recovered by per- O -methylated ,-cyclodextrin. No irreversible denaturation of ChT occurred upon binding with FeP8M. The kinetic data support the observation that, for nonincubated samples, both inhibitors did not cause significant conformational change in ChT and inhibited the ChT activity by covering the active site of the enzyme. [source] Influence of Ionization State on the Activation of Temocapril by hCES1: A Molecular-Dynamics StudyCHEMISTRY & BIODIVERSITY, Issue 11 2009Giulio Vistoli Abstract Temocapril is a prodrug whose hydrolysis by carboxylesterase 1 (CES1) yields the active ACE inhibitor temocaprilat. This molecular-dynamics (MD) study uses a resolved structure of the human CES1 (hCES1) to investigate some mechanistic details of temocapril hydrolysis. The ionization constants of temocapril (pK1 and pK3) and temocaprilat (pK1, pK2, and pK3) were determined experimentally and computationally using commercial algorithms. The constants so obtained were in good agreement and revealed that temocapril exists mainly in three ionic forms (a cation, a zwitterion, and an anion), whereas temocaprilat exists in four major ionic forms (a cation, a zwitterion, an anion, and a dianion). All these ionic forms were used as ligands in 5-ns MS simulations. While the cationic and zwitterionic forms of temocapril were involved in an ion-pair bond with Glu255 suggestive of an inhibitor behavior, the anionic form remained in a productive interaction with the catalytic center. As for temocaprilat, its cation appeared trapped by Glu255, while its zwitterion and anion made a slow departure from the catalytic site and a partial egress from the protein. Only its dianion was effectively removed from the catalytic site and attracted to the protein surface by Lys residues. A detailed mechanism of product egress emerges from the simulations. [source] How Does a Membrane Protein Achieve a Vectorial Proton Transfer Via Water Molecules?CHEMPHYSCHEM, Issue 18 2008Steffen Wolf Abstract We present a detailed mechanism for the proton transfer from a protein-bound protonated water cluster to the bulk water directed by protein side chains in the membrane protein bacteriorhodopsin. We use a combined approach of time-resolved Fourier transform infrared spectroscopy, molecular dynamics simulations, and X-ray structure analysis to elucidate the functional role of a hydrogen bond between Ser193 and Glu204. These two residues seal the internal protonated water cluster from the bulk water and the protein surface. During the photocycle of bacteriorhodopsin, a transient protonation of Glu204 leads to a breaking of this hydrogen bond. This breaking opens the gate to the extracellular bulk water, leading to a subsequent proton release from the protonated water cluster. We show in detail how the protein achieves vectorial proton transfer via protonated water clusters in contrast to random proton transfer in liquid water. [source] Protein,Protein Interactions in Complex Cosolvent Solutions,CHEMPHYSCHEM, Issue 5 2007Nadeem Javid Abstract The effects of various kosmotropic and chaotropic cosolvents and salts on the intermolecular interaction potential of positively charged lysozyme is evaluated at varying protein concentrations by using synchrotron small-angle X-ray scattering in combination with liquid-state theoretical approaches. The experimentally derived static structure factors S(Q) obtained without and with added cosolvents and salts are analysed with a statistical mechanical model based on the Derjaguin,Landau,Verwey,Overbeek (DLVO) potential, which accounts for repulsive and attractive interactions between the protein molecules. Different cosolvents and salts influence the interactions between protein molecules differently as a result of changes in the hydration level or solvation, in charge screening, specific adsorption of the additives at the protein surface, or increased hydrophobic interactions. Intermolecular interaction effects are significant above protein concentrations of 1 wt,%, and with increasing protein concentration, the repulsive nature of the intermolecular pair potential V(r) increases markedly. Kosmotropic cosolvents like glycerol and sucrose exhibit strong concentration-dependent effects on the interaction potential, leading to an increase of repulsive forces between the protein molecules at low to medium high osmolyte concentrations. Addition of trifluoroethanol exhibits a multiphasic effect on V(r) when changing its concentration. Salts like sodium chloride and potassium sulfate exhibit strong concentration-dependent changes of the interaction potential due to charge screening of the positively charged protein molecules. Guanidinium chloride (GdmCl) at low concentrations exhibits a similar charge-screening effect, resulting in increased attractive interactions between the protein molecules. At higher GdmCl concentrations, V(r) becomes more repulsive in nature due to the presence of high concentrations of Gdm+ ions binding to the protein molecules. Our findings also imply that in calculations of thermodynamic properties of proteins in solution and cosolvent mixtures, activity coefficients may not generally be neglected in the concentration range above 1 wt,% protein. [source] A Colloidal Au Monolayer Modulates the Conformation and Orientation of a Protein at the Electrode/Solution InterfaceCHEMPHYSCHEM, Issue 8 2005Xiue Jiang Abstract The orientation and conformation of adsorbed cytochrome c (cyt c) at the interface between an electrode modified with colloidal Au and a solution were studied by electrochemical, spectroscopic, and spectroelectrochemical techniques. The results indicate that the colloidal Au monolayer formed via preformation of an organic self-assembled monolayer (SAM) can increase the electronic coupling between the SAM and cyt c in the same manner as bifunctional molecular bridges, one functional group of which is bound to the electrode surface while the other interacts with the protein surface. The approach of cyt c to the modified electrode/solution interface can be assisted by strong interactions of the intrinsic charge of colloidal particles with cyt c, while the heme pocket remains almost unchanged due to the screening effect of the negatively charged field created by the intrinsic charge. The conformational changes of cyt c induced by its adsorption at a bare glassy carbon electrode/solution interface and the effect of the electric field on the ligation state of the heme can be avoided at the colloidal-Au-modified electrode/solution interface. Finally, a possible model for the adsorption orientation of cyt c at the colloidal-Au-modified electrode/solution interface is proposed. [source] The effect of HAMP domains on class IIIb adenylyl cyclases from Mycobacterium tuberculosisFEBS JOURNAL, Issue 12 2004Jürgen U. Linder The genes Rv1318c, Rv1319c, Rv1320c and Rv3645 of Mycobacterium tuberculosis are predicted to code for four out of 15 adenylyl cyclases in this pathogen. The proteins consist of a membrane anchor, a HAMP region and a class IIIb adenylyl cyclase catalytic domain. Expression and purification of the isolated catalytic domains yielded adenylyl cyclase activity for all four recombinant proteins. Expression of the HAMP region fused to the catalytic domain increased activity in Rv3645 21-fold and slightly reduced activity in Rv1319c by 70%, demonstrating isoform-specific effects of the HAMP domains. Point mutations were generated to remove predicted hydrophobic protein surfaces in the HAMP domains. The mutations further stimulated activity in Rv3645 eight-fold, whereas the effect on Rv1319c was marginal. Thus HAMP domains can act directly as modulators of adenylyl cyclase activity. The modulatory properties of the HAMP domains were confirmed by swapping them between Rv1319c and Rv3645. The data indicate that in the mycobacterial adenylyl cyclases the HAMP domains do not display a uniform regulatory input but instead each form a distinct signaling unit with its adjoining catalytic domain. [source] | ||||||||||||||||||||||||||||