An evolution-based analysis scheme to identify CO2/O2 specificity-determining factors for ribulose 1,5-bisphosphate carboxylase/oxygenase

Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCo) catalyzes a rate-limiting step in photosynthetic carbon assimilation (reacting with CO2) and its competitive photo-respiratory carbon oxidation (reacting with O2). RuBisCo enzyme with an enhanced CO2/O2 specificity would boost the ability to make great progress in agricultural production and environmental management. RuBisCos in marine non-green algae, resulting from an earlier endo-symbiotic event, diverge greatly from those in green plants and cyanobacteria and, further, have the highest CO2/O2 specificity whereas RuBisCos in cyanobacteria have the lowest. We assumed that there exist different levels of CO2/O2 specificity-determining factors, corresponding to different evolutionary events and specificity levels. Based on this assumption, we devised a scheme to identify these substrate-determining factors. From this analysis, we are able to discover different categories of the CO2/O2 specificity-determining factors that show which residue substitutions account for (relatively) small specificity changes, as happened in green plants, or a tremendous enhancement, as observed in marine non-green algae. Therefore, the analysis can improve our understanding of molecular mechanisms in the substrate specificity development and prioritize candidate specificity-determining surface residues for site-directed mutagenesis.     

Dimeric character of a basic phospholipase A2 from cobra venom: experimental and modelling study

When it is gel filtered on Sephadex in the absence of calciumions, basic phospholipase A₂ from Naja nigricollis venom elutesas a dimer. In order to study the possibility of this dimerizationfrom a structural point of view, three-dimensional models ofboth monomeric and dimeric N. nigricollis phospholipases A havebeen graphically built on the basis of homologies with the phospholipasesA₂ from pancreatic bovine and Crotalus atrox venom. The buildingof a duneric model is made possible by the deletion of a particularloop of the bovine structure. The predicted models of N. nigricollisphospholipase A₂ have been checked using molecular mechanicsand molecular dynamics techniques according to a suitable protocolwhich has been developed starting from refined X-ray structuresof phospholipases A₂ as the test case. The observed stabilityof the dimeric model, in the absence of calcium, agrees withthe hypothesis of the dimerization of the basic phospholipaseA Particularly, Arg31, which replaces the hydrophobic residuepresent in pancreatic bovine and C.atrox venom phospholipasesA₂, contributes to this stability.     

Improving solubility and refolding efficiency of human V(H)s by a novel mutational approach

The antibody V(H) domains of camelids tend to be soluble and to resist aggregation, in contrast to human V(H) domains. For immunotherapy, attempts have therefore been made to improve the properties of human V(H)s by camelization of a small set of framework residues. Here, we have identified through sequence comparison of well-folded llama V(H) domains an alternative set of residues (not typically camelid) for mutation. Thus, the solubility and thermal refolding efficiency of a typical human V(H), derived from the human antibody BT32/A6, were improved by introduction of two mutations in framework region (FR) 1 and 4 to generate BT32/A6.L1. Three more mutations in FR3 of BT32/A6.L1 further improved the thermal refolding efficiency while retaining solubility and cooperative melting profiles. To demonstrate practical utility, BT32/A6.L1 was used to construct a phage display library from which were isolated human V(H)s with good antigen binding activity and solubility. The engineered human V(H) domains described here may be useful for immunotherapy, due to their expected low immunogenicity, and in applications involving transient high temperatures, due to their efficient refolding after thermal denaturation.     

Comparison of the construction of a 3-D model for human thromboxane synthase using P450cam and BM-3 as templates: implications for the substrate binding pocket

A 3-D model of human thromboxane A₂ synthase (TXAS) was constructedusing a homology modeling approach based on information fromthe 2.0 crystal structure of the hemoprotein domains of cytochromeP450BM-3 and P450cam. P450BM-3 is a bacterial fatty acid monooxygenaseresembling eukaryotic microsomal cytochrome P450s in primarystructure and function. TXAS shares 26.4% residue identity and48.4% residue similarity with the P450BM-3 hemoprotein domain.The homology score between TXAS and P450BM-3 is much higherthan that between TXAS and P450cam. Alignment between TXAS andthe P450BM-3 hemoprotein domain or P450cam was determined throughsequence searches. The P450BM-3 or P450cam main-chain coordinateswere spplied to the TXAS main chain in those sements where thetwo sequences were well aligned. These segments were linkedto one another using a fragment search method, and the sidechains were added to produce a 3-D model for TXAS. A TXAS substrate,prostaglandin H₂ (PGH₂) was docked into the TXAS cavity correspondingto the arachidonic acid binding pocket in P450BM-3 or camphorbinding site in P450cam. Regions of the heme and putative PGH2binding cavities in the TXAS model were identified and analyzed.The segments and residues involved in the active-site pocketof the TXAS model provide reasonable candidates for TXAS proteinengineering and inhibitor design. Comparison of the TXAS modelbased on P450BM-3 with another TXAS model based on the P450BM-3with another TXAS model based on the P450cam structure indicatedthat P450BM-3 is a more suitable template for homology modelingof TXAS.     

Site-directed mutagenesis and X-ray crystallography of two phospholipase A2 mutants: Y52F and Y73F

Tyr52 and Tyr73 are conserved amino acid residues throughoutall vertebrate phospholipases A₂. They are part of an extendedhydrogen bonding system that links the N-terminal -NH⁺₃ -groupto the catalytic residues His48 and Asp99. These tyrosines werereplaced by phenylalanines in a porcine pancreatic phospholipaseA₂ mutant, in which residues 62–66 had been deleted (62–66PLA₂).The mutations did not affect the catalytic properties of theenzyme, nor the folding kinetics. The stability against denaturatlonby guanidine hydrochloride was decreased, however. To analysehow the enzyme compensates for the loss of the tyrosine hydroxylgroup, the X-ray structures of the Y52F and AY73F mutants weredetermined. After crystallographic refinement the final crystallographicR-factors were 18.1% for the %Y52F mutant (data between 7 and2.3 Å resolution) and 19.1% for the Y73F mutant (databetween 7 and 2.4 Å resolution). No conformational changesoccurred in the mutants compared with the 62–66PLA₂, butan empty cavity formed at the site of the hydroxyl group ofthe former tyrosine. In both mutants the Asp99 side chain losesone of its hydrogen bonds and this might explain the observeddestabilization.     

Enhancing the stability of microsomal cytochrome b5: a rational approach informed by comparative studies with the outer mitochondrial membrane isoform

The outer mitochondrial membrane isoform of mammalian cytochrome b5 (OM b5) is much less prone to lose heme than the microsomal isoform (Mc b5), with a conserved difference at position 71 (leucine versus serine) playing a major role. We replaced Ser71 in Mc b5 with Leu, with the prediction that it would retard heme loss by diminishing polypeptide expansion accompanying rupture of the histidine to iron bonds. The strategy was partially successful in that it slowed dissociation of heme from its less stable orientation in bMc b5 (B). Heme dissociation from orientation A was accelerated to a similar extent, however, apparently owing to increased binding pocket dynamic mobility related to steric strain. A second mutation (L32I) guided by results of previous comparative studies of Mc and OM b5s diminished the steric strain, but much greater relief was achieved by replacing heme with iron deuteroporphyrin IX (FeDPIX). Indeed, the stability of the Mc(S71L) b5 FeDPIX complex is similar to that of the FeDPIX complex of OM b5. The results suggest that maximizing heme binding pocket compactness in the apo state is a useful general strategy for increasing the stability of engineered or designed proteins.     

Adaptability of restrained molecular dynamics for tertiary structure prediction: application to Crotalus atrox venom phospholipase A2

In order to assess the adaptability and/or applicability ofthe restrained molecular dynamics (RMD) simulation for buildinga possible tertiary structure of a protein from the X-ray crystalstructure of a family reference protein, the tertiary structureprediction of Crotalus atrox venom phospholipase A₂ (PLA₂) wasattempted based on the X-ray crystal structure of bovine pancreaticPLA₂. For the formation of secondary and tertiary structuresfrom the fully extended starting structure, the RMD simulationwith interatomic distance restraints and torsion angle restraints,which were derived from homologous amino acid sequence regionsin the reference protein, was carried out until the molecularsystem was fully equilibrated. The predicted tertiary structureof C.atrox venom PLA₂ was compared with its X-ray crystal structure,and furthermore the utility of this method was discussed byreference to the similar tertiary structure prediction of ß-trypsinfrom the X-ray crystal structure of an elastase.     

Influenza virus M2 protein: a molecular modelling study of the ion channel

The influenza A M₂ protein forms cation-selective ion channelswhich are blocked by the anti-influenza drug amantadine. A molecularmodel of the M₂ channel is presented in which a bundle of fourparallel M₂ transbilayer helices surrounds a central ion-permeablepore. Analysis of helix amphipathicity was used to aid determinationof the orientation of the helices about their long axes. Thehelices are tilted such that the N-terminal mouth of the poreis wider than the C-terminal mouth. The channel is lined byresidues V27, S31 and I42. Residues D24 and D44 are locatedat opposite mouths of the pore, which is narrowest in the vicinityof I42. Energy profiles for interaction of the channel withNa⁺, amantadine-H⁺ and cyclopentylamine-H⁺ are evaluated. Theinteraction profile for Na⁺ exhibits three minima, one at eachmouth of the pore, and one in the region of residue S31. Theamantadine-H⁺ profile exhibits a minimum close to S31 and abarrier near residue I42. This provides a molecular model foramantadine-H⁺ block of M₂ channels. The profile for cyclopentylamine-H⁺does not exhibit such a barrier. It is predicted that cyclopentyl-amine-H⁺will not act as an M₂ channel blocker.     

Influence of size and polarity of residue 31 in porcine pancreatic phospholipase A2 on catalytic properties

Residue 31 of porcine pancreatic phospholipase A₂ (PLA₂) islocated at the entrance to the active site. To study the roleof residue 31 in PLA₂, six mutant enzymes were produced by site-directedmutagenesis, replacing Leu by either Trp, Arg, Ala, Thr, Seror Gly. Direct binding studies indicated a three to six timesgreater affinity of the Trp31 PLA₂ for both monomeric and micellarsubstrate analogs, relative to the wild-type enzyme. The otherfive mutants possess an unchanged affinity for monomers of theproduct analog n-decylphosphocholine and for micelles of thediacyl substrate analog rac-l,2-dioctanoylamino-dideoxy-glycero-3-phosphocholine.The affinities for micelles of the monoacyl product analog n-hexadecylphosphocholinewere decreased 9–20 times for these five mutants. Kineticstudies with monomeric substrates showed that the mutants haveV_max values which range between 15 and 70% relative to the wild-typeenzyme. The V_max values for micelles of the zwitterionic substratel,2-dioctanoyl-sn-glycero-3-phosphocholine were lowered 3–50times. The K_m values for the monomeric substrate and the k_mvalues for the micellar substrate were hardly affected in thecase of five of the six mutants, but were considerably decreasedwhen Trp was present at position 31. The results of these investigationspoint to a versatile role for the residue at position 31: involvementin the binding and orientating of monomeric substrate (analogs),involvement in the binding of the enzyme to micellar substrateanalogs and possibly involvement in shielding the active sitefrom excess water.     

Function of the fully conserved residues Asp99, Tyr52 and Tyr73 in phospholipase A2

In the active centre of pancreatic phospholipase A₂ His48 isat hydrogen-bonding distance to Asp99. This Asp-His couple isassumed to act together with a water molecule as a catalytictriad. Asp99 is also linked via an extended hydrogen bondingsystem to the side chains of Tyr52 and Tyr73. To probe the functionof the fully conserved Asp99, Tyr52 and Tyr73 residues in phospholipaseA₂, the Asp99 residue was replaced by Asn, and each of the twotyrosines was separately replaced by either a Phe or a Gln.The catalytic and binding properties of the Phe52 and Phe73mutants did not change significantly relative to the wild-typeenzyme. This rules out the possibility that either one of thetwo Tyr residues in the wild-type enzyme can function as anacyl acceptor or proton donor in catalysis. The Gln73 mutantcould not be obtained in any significant amounts probably dueto incorrect folding. The Gln52 mutant was isolated in low yield.This mutant showed a large decrease in catalytic activity whileits substrate binding was nearly unchanged. The results suggesta structural role rather than a catalytic function of Tyr52and Tyr73. Substitution of asparagine for aspartate hardly affectsthe binding constants for both monomeric and micellar substrateanalogues. Kinetic characterization revealed that the Asn99mutant has retained no less than 65% of its enzymatic activityon the monomeric substrate rac 1,2-dihexanoyldithio-propyl-3-phosphocholine,probably due to the fact that during hydrolysis of monomericsubstrate by phospholipase A₂ proton transfer is not the rate-limitingstep. The Asp to Asn substitution decreases the catalytic rateon micellar 1,2-dioctanoyl-sn-glycero-3-phosphocholine 25-fold.To explain this remaining activity we suggest that in the mutantthe Asn99 orients His48 in the same way as Asp99 orients His48in native phospholipase A₂ and that the lowered activity iscaused by a reduced stabilization of the transition state.     

A three-dimensional construction of the active site (region 507749) of human neutral endopeptidase (EC.3.4.24.11)

A three-dimensional model of the 507–749 region of neutralendopeptidase-24.11 (NEP; E.C.3.4.24.11) was constructed integratingthe results of secondary structure predictions and sequencehomologies with the bacterial endopeptidase thermolysin. Additionaldata were extracted from the structure of two other metalloproteases,astacin and stromelysin. The resulting model accounts for themain biological properties of NEP and has been used to describethe environment close to the zinc atom defining the catalyticsite. The analysis of several thiol inhibitors, complexed inthe model active site, revealed the presence of a large hydrophobicpocket at the S₁' subsite level. This is supported by the natureof the constitutive amino acids. The computed energies of boundinhibitors correspond with the relative affinities of the stereoisomersof benzofused macrocycle derivatives of thiorphan. The modelcould be used to facilitate the design of new NEP inhibitors,as illustrated in the paper.     

Engineering cytochrome P-450cam to increase the stereospecificity and coupling of aliphatic hydroxylation

Site-directed mutants were constructed in cytochrome P-450_camto re-engineer the stereochemistry and coupling of ethylbenzenehydroxyiation. The reaction with wild-type (WT) enzyme producesone regioisomer 1-phenylethanol with 5% reduced nicotinamideadenine deoxyribonucleic acid to product conversion of and aratio of 73:27 for the R and S enantiomers respectively. Ethyibenzenewas modeled into the active site of WT P-450_cam in a rigid modeand oriented to optimize either pro-R or pro-S hydrogen abstraction.Residues T101, T185 and V247 make extensive contacts with thesubstrate in the static complexes and were therefore chosenfor site-directed mutagenesis. Single mutants T101M, V247A andV247M are more stereospedik producing 89,87 and 82% (R)-1-phenylethanolrespectively. The coupling of the reaction is doubled for thesingle mutants T185L, T185F and V247M. In an effort to engineerincreased stereospecificity and coupling into a single catalystthe T101M, T185F and V247M mutants were combined in a multiplemutant of P-450_cam.This protein hydroxylates ethyibenzene resultingin an R:S ratio of 87:13 for the 1-phenylethanols and 13% couplingof reducing equivalents to product. The catalytic stereospecificityand stoichiometry with T101M–T185F–V247M does notrepresent a summation of the changes observed for the singlemutants. A portion of the individual effects on substrate recognitionproduced by the single substitutions is either eliminated ordegenerate within the triple mutant.     

Crystallization and structure solution at 4 A resolution of the recombinant synthase domain of N(5'-phosphoribosyl)anthranilate isomerase:indole-3-glycerol-phosphate synthase from Escherichia coli complexed to a substrate analogue

The recombinant synthase domain of the bifunctional enzyme N-(5'-phosphoribosyl)anthranilateisomerase:indole-3-glycerol-phosphate synthase from Escherichiacoli has been crystallized, and the structure has been solvedat 4 Å resolution. Two closely related crystal forms grownfrom ammonium sulphate diffract to 2 Å resolution. Oneform (space group R32, a = 163 Å, = 29.5°) containsthe unliganded synthase domain; the second crystal form (spacegroup P6₃22, a = 144 Å, c = 158 Å) is co-crystallizedwith the substrate analogue N-(5'-phosphoribit-1-yl)anthranilate.The structure of the synthase–inhibitor complex has beensolved by the molecular replacement method. This achievementrepresents the first successful use of a (ß)_g-barrelmonomer as a trial model. The recombinant synthase domain associatesas a trimer in the crystal, the molecules being related by apseudo-crystallographic triad. The interface contacts betweenthe three domains are mediated by those residues that are alsoinvolved in the domain interface of the bifunctional enzyme.This system provides a model for an interface which is usedin both intermolecular and intramolecular domain contacts.     

Improvement of turn structure prediction by molecular dynamics: a case study of {alpha}1-purothionin

Because of the problems in predicting a correct conformationfor loop regions in homology-based prediction, disagreementsare often found between the predicted models and the refinedX-ray structures of the same protein in loop regions. Such asituation has been encountered for ₁-purothionin (₁-PT). Hence,attempts have been made to improve the predicted model of ₁PTby limited molecular dynamics using both AMBER and XPLOR. Withmolecular dynamics, the previously predicted incorrect turnregion reverts to the correct conformation as seen in the X-rayrefined structure. In contrast to the model which is not subjectedto molecular dynamics, the improved model refines with the X-raydata of ₁PT in fewer cycles, without any manual rebuilding andwith comparable or better refinement statistics. Also, the improvedmodel serves as a better starting model in the determinationof the structure with the molecular replacement methods.     

Thermitase and proteinase K: a comparison of the refined three-dimensional structures of the native enzymes

We compare the three-dimensional structures of thermitase andof proteinase K determined by X-ray crystallography to a resolutionof 1.4 and 1.48 Å respectively. Both enzymes are relativelystable towards heat and denaturating agents and are representativeof a subgroup of subtilisins characterized by a free SH groupclose to the active site histidine. Even though they have lowsequence homology, the overall tertiary structures are highlyconserved. The high resolution structures are compared in termsof the overall fold of the molecules, the active sites, thecalcium binding sites, disulphide bridge positions, the positionsof the charged residues and the solvent structure. Most subtilisinssuch as thermitase are of prokaryotic origin and proteinaseK is up to now the only known eukaryotic structure.     

A new approach to artificial and modified proteins: theory-based design, synthesis in a cell-free system and fast testing of structural properties by radiolabels

A novel approach to the creation of artificial and modifiedproteins has been elaborated. The approach includes a sequencedesign based on the molecular theory of protein secondary structureand folding patterns, gene expression in a cell-free systemand testing of structural properties of the synthesized polypeptidesat a nanogram level using radiolabelled chains. The approachhas been applied to a new synthetic protein albebetin whichhas been designed to form a 3-D fold which does not contradictany structural rule but has been never observed up to now innatural proteins. Using size-exclusion chromatography, urea-gradientelectrophoresis and limited proteolysis of a radiolabelled chain,it has been shown that the artificial protein is nearly as compactas natural proteins, cooperatively unfolds at high urea concentrationsand has some structural features of a definite structure consistentwith the designed one. As albebetin has been designed as consistingof two structural repeats, a ‘halfalbebetin’ (oneof these repeats) has also been synthesized and studied. Itwas shown that ‘half-albebetin’ is also compact     

Inhibition of cytotoxicity and amyloid fibril formation by a D-amino acid peptide that specifically binds to Alzheimer's disease amyloid peptide

Alzheimer's disease (AD) is a progressive neurodegenerative disorder. The 'amyloid cascade hypothesis' assigns the amyloid-beta-peptide (Abeta) a central role in the pathogenesis of AD. Although it is not yet established, whether the resulting Abeta aggregates are the causative agent or just a result of the disease progression, polymerization of Abeta has been identified as a major feature during AD pathogenesis. Inhibition of the Abeta polymer formation, thus, has emerged as a potential therapeutic approach. In this context, we identified peptides consisting of d-enantiomeric amino acid peptides (d-peptides) that bind to Abeta. D-peptides are known to be more protease resistant and less immunogenic than the respective L-enantiomers. Previously, we have shown that a 12mer D-peptide specifically binds to Abeta amyloid plaques in brain tissue sections from former AD patients. In vitro obtained binding affinities to synthetic Abeta revealed a K(d) value in the submicromolar range. The aim of the present study was to investigate the influence of this d-peptide to Abeta polymerization and toxicity. Using cell toxicity assays, thioflavin fluorescence, fluorescence correlation spectroscopy and electron microscopy, we found a significant effect of the d-peptide on both. Presence of D-peptides (dpep) reduces the average size of Abeta aggregates, but increases their number. In addition, Abeta cytotoxicity on PC12 cells is reduced in the presence of dpep.     

Mix'n'Match: an improved multiple sequence alignment procedure for distantly related proteins using secondary structure predictions, designed to be independent of the choice of gap penalty and scoring matrix

A new multiple sequence alignment procedure is presented. Severaldifferent multiple alignments are made using differing criteria.Having divided the sequences into strongly conserved regions(SCRs) and loosely conserved regions (LCRs), the ‘best’alignment for each LCR is chosen, independently of the otherLCRs, from a selection of possibilities in the multiple alignments.To help make this choice for each LCR, the secondary structureis predicted and shown alongside each different possible alignment.One advantage of this method over automatic, non-interactivemethods, is that the final alignment is not dependent on thechoice of a single set of scoring parameters. Another is that,by allowing interactive choice and by taking account of secondarystructural information, the final alignment is based more onbiological rather than mathematical factors. This method canproduce better alignments than any of the initial automaticmultiple alignment methods used.     

Tackling increased CO2 concentration in feeds for existing acid gas removal units: A simulation study based on a customized CO2 kinetics model

A Middle East-based amine sweetening unit, with an overall capacity of about 2.2 BSCFD of gas, is among the world’s largest process plants and currently processes sour gas with 10 mol% of hydrogen sulfide (H₂S) and carbon dioxide (CO₂) put together. Current expectation is that acid gas contents in the feed may increase beyond the design limit of the plant. The present work is an effort to quantify the effects of the feed gas CO₂ increase on the plant and to proffer solutions to handle these effects efficiently. We revised the kinetics of amine-based CO₂ absorption correlation of an existing model using real-data-driven parameters re-estimation. Evolutionary technique that employs particle swarm optimization algorithm is used for this purpose. The new CO₂ kinetic model is inserted in a first-principle process simulator, ProMax® V4.0, in order to analyze various solutions necessary to mitigate the operational challenges due to increased feed CO₂. The process plant with present design and operating conditions is determined to handle up to 8.45 mol% CO₂ contents in the sour gas feed. Further results revealed that methyldiethanolamine, diethanolamine, and dimethyl ether propylene glycol (DEPG) could not handle this high feed CO₂ challenge, even at maximum (design) steam and solvent usage. However, diglycolamine exclusively renders the solution as it treats high CO₂ feed gas efficiently with allowable utility consumption, while satisfying the constraints imposed by product gas specifications.     

Mechanistic understanding of CO2-induced plasticization of a polyimide membrane: A combination of experiment and simulation study

Experimental measurements and fully atomistic simulations are carried out to examine the CO₂-induced plasticization of a polyimide membrane synthesized from 4,4′-(hexafluoroisopropylidene) diphthalic anhydride (6FDA) and 4,4′-oxydianiline (ODA). With increasing feed pressure, the permeability of CO₂ in the 6FDA-ODA membrane initially decreases, crosses a minimum, and then increases. The plasticization pressure is estimated to be at approximately 8 atm. The radial distribution functions between CO₂ and polyimide atoms reveal that the imide groups are the preferential sorption sites, followed by the ether and CF₃ groups. The experimental and simulated sorption isotherms of CO₂ are in fairly good agreement. At low loadings, CO₂ molecules are largely trapped with small mobility. With increasing loading, the polyimide membrane exhibits a depressed glass transition temperature, a dilated volume and an increased fractional free volume. In addition, larger and more interconnected voids appear and the mean radius of voids increases from 2.5 to 3.3 Å with increasing CO₂ loading. Consequently, the mobility of both CO₂ molecules and polymer chains is enhanced. Based on molecular displacement, the percentages of three types of motions (jumping, trapped, and continuous) are estimated for CO₂ in the membrane. The continuous motion contributes predominantly to CO₂ diffusion. At a high loading, the ether groups in the polyimide chains exhibit a significant effect on plasticization. It is therefore suggested that the plasticization could be suppressed by substituting the ether groups. The microscopic information of this study is particularly useful for the quantitative understanding of plasticization.