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1.
The crystallographic structures of jack bean canavalin and Frenchbean phaseolin have been used to construct a homology modelof the storage vicilin of cocoa. Reported molecular weightsfor cocoa storage protein subunits correlate with proteolysisat the site of a large hydrophilic insert in the mature protein.Burial of the hydrophobic amino acids on trimer formation isa strongly conserved feature in the vicilin family. Histidineresidues also sit at the monomer-monomer interfaces of the trimerand are likely to contribute to the decreased solubility ofcocoa vicilin at mild acidic pH, which is generally consideredto be caused solely by aggregation near to the isoelectric point.Electrostatic calculations suggest that such an arrangementof histidine residues in the absence of specific counterionbinding will not favour the particular geometry of trimer formationbelow neutral pH. Higher order aggregates that do not excludehistidine charge from the solvent may be favoured, aiding theprecipitation of cocoa vicilin at mild acidic pH. This suggestionis considered for the vicilin family. The hypothesis could contributeto an understanding of the pH and ionic strength dependenceof vicilin solubility in vitro, and possibly of the behaviourof vicilins in the seed storage environment 相似文献
2.
Fersht Alan R.; Sternberg Michael J.E. 《Protein engineering, design & selection : PEDS》1989,2(7):527-530
The relationship between the effective dielectric constant thatmodels the electrostatic effect from a charged side chain ina protein was evaluated both experimentally and theoretically.Experimental values were obtained from the shifts in pKa thatresulted from point mutations of side chains in subtilisin.Theoretical values were obtained from an iterative solutionto Poisson's equation that considers the dielectric responseof the protein and the solvent together with charge positions.There is no simple relationship between the effective dielectricconstant and the distance from the charge responsible for theinteractions. For some charge positions a linear but not a directproportional relationship of the effective dielectric with distanceof separation was observed. Thus, simple models such as a lineardistance-dependence for the dielectric response are not suitableto evaluate electrostatic effects in proteins. 相似文献
3.
Arima J Uesugi Y Iwabuchi M Hatanaka T 《Protein engineering, design & selection : PEDS》2008,21(1):45-53
We purified and characterized the aminopeptidase P from Streptomyces costaricanus TH-4 (thAPP). This enzyme has a tetramer structure, a metal-ion preference toward Zn, broad substrate specificity and a narrow pH dependency for activity. The primary structure of thAPP, respectively, exhibits 91% and 65% identity with those of two other APPs-APP I and APP II-from Streptomyces lividans (slAPP I and slAPP II). We next overexpressed the genes encoding thAPP and slAPP II in Escherichia coli and characterized them. Two differences were apparent in their properties: slAPP II formed a dimer, whereas thAPP formed a tetramer; also, the alkaline side pKa for the catalytic action of slAPP II is higher than that of thAPP. Investigation using chimeras of both enzymes revealed that the N-terminal domain is associated with the determination of pKa values for catalytic action and quaternary structure. 相似文献
4.
Molecular modelling of xylose isomerase catalysis: the role of electrostatics and charge transfer to metals 总被引:1,自引:0,他引:1
Fuxreiter Monika; Farkas Odon; Naray-Szabo Gabor 《Protein engineering, design & selection : PEDS》1995,8(9):925-933
The two main steps of the mechanism of xylose-xylulose conversioncatalysed by D-xylose isomerase, the ring opening of xyloseand the isomerization of the opened product by hydride transfer,were investigated by molecular mechanical and molecular orbitaltechniques. The activation energies calculated for these reactionsclearly showed that hydrogen transfer is the rate-determiningstep of the enzymatic isomerization and that Mg2+ ions activatewhereas Zn2+ ions inhibit the reaction, in agreement with theexperiments. The remarkable differences between the net chargesof these ions found by molecular orbital calculations and theinspection of the protein electrostatic potential around thereaction intermediates indicate that the main role of bivalentmetal ions should be the electrostatic stabilization of thesubstrate transition states. In order to propose a more detailedmechanism, an attempt was made to clarify the effects of nearbyresidues (e.g. His54, Asp57, Lysl83, Asp257) in the reaction.Different isomerization mechanisms, such as through an enediolintermediate, were examined and could be excluded, in additionto the charge-relay mechanism during the ring opening. 相似文献
5.
The locations of the cations bound to the peptide group at theC-termini and the anions attached to the main-chain NH groupat the N-termini of helices are analysed. The ions are hardlyfound along the helical axis, where the effect due to the helixmacrodipole is likely to be the maximum. The disposition ofthe ions appears to be controlled more by the stereoelectronicrequirements of the ligand group rather than any long distanceelectric field. This and other related structural observationscall for some circumspection in assigning a role for the helixdipole in protein structure and function. 相似文献
6.
Sun Ying-Chieh; Veenstra David L.; Kollman Peter A. 《Protein engineering, design & selection : PEDS》1996,9(3):273-281
Free energy calculations were carried out to determine the relativeunfolding free energy of the Ile96 wild type and Ala96 mutantbarnases. The total calculated free energies suggest that substitutionof Ile96 with Ala destabilizes barnase by 3.9 kcal/mol, whichis in good agreement with the independently determined experimentalvalues of 4.0 and 3.3 kcal/mol and a previous simulation. However,a decomposition of the free energy finds the dominant contributionsto this free energy arising from the noncovalent Interactionsbetween the perturbed group and distant residues of barnasein the sequence and water molecules and only a very small contributionfrom covalent interactions. This is in contrast to the previoussimulation, using the dual topology methodology, which produceda decomposition with an {small tilde}60% free energy contributionfrom changes in covalent interactions. The use of the singletopology employed in the present calculations and the dual topologyemployed in the previous study are analyzed in order to understandthe contrast between the present results and the results ofthe previous study. 相似文献
7.
Tanha J Nguyen TD Ng A Ryan S Ni F Mackenzie R 《Protein engineering, design & selection : PEDS》2006,19(11):503-509
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. 相似文献
8.
Muskal Steven M.; Holbrook Stephen R.; Kim Sung-Hou 《Protein engineering, design & selection : PEDS》1990,3(8):667-672
The bonding states of cysteine play important functional andstructural roles in proteins. In particular, disulfide bondformation is one of the most important factors influencing thethree-dimensional fold of proteins. Proteins of known structurewere used to teach computer-simulated neural networks rulesfor predicting the disulfide-bonding state of a cysteine givenonly its flanking amino acid sequence. Resulting networks makeaccurate predictions on sequences different from those usedin training, suggesting that local sequence greatly influencescysteines in disulfide bond formation. The average predictionrate after seven independent network experiments is 81.4% fordisulfide-bonded and 80.0% for non-disulfide-bonded scenarios.Predictive accuracy is related to the strength of network outputactivities. Network weights reveal interesting position-dependentamino acid preferences and provide a physical basis for understandingthe correlation between the flanking sequence and a cysteine'sdisulfide-bonding state. Network predictions may be used toincrease or decrease the stability of existing disulfide bondsor to aid the search for potential sites to introduce new disulfidebonds. 相似文献
9.
Bohm Gerald; Muhr Rudolf; Jaenicke Rainer 《Protein engineering, design & selection : PEDS》1992,5(3):191-195
A new method based on neural network theory is presented toanalyze and quantify the information content of far UV circulardichroism spectra. Using a backpropagation network model witha single hidden layer between input and output, it was possibleto deduce five different secondary structure fractions (helix,parallel and antiparallel ß-sheet, ß-turnand random coil) with satisfactory correlations between calculatedand measured secondary structure data. We demonstrate that foreach wavelength interval a specific network is suitable. Theremaining discrepancy between the secondary structure data fromneural network prediction and crystallography may be attributedto errors in the determination of protein concentration andrandom noise in the CD signal, as indicated by simulations. 相似文献
10.
A simple electrostatic model has been used to investigate theextent to which the structure of protein molecules is organizedto optimize the internal electrostatic interactions. We findthat the model provides a favorable total intra-protein electrostaticenergy for almost all polar and charged groups of atoms, suggestinga high degree of structural optimization. By contrast, a significantfraction of individual groupgroup interactions are foundto be unfavorable. An analysis as a function of the range ofinteractions included shows the electrostatic organization isgenerally relatively short range (up to 6 or 7 Å betweengroup centers). Although the model is very simple, it is usefulfor assessing the overall quality of protein experimental structures,for pin-pointing some types of errors and as a guide to improvingprotein design. 相似文献
11.
Molecular dynamics of the 5-HT1a receptor and ligands 总被引:2,自引:0,他引:2
Sylte Ingebrigt; Edvardsen Oyvind; Dahl Svein G. 《Protein engineering, design & selection : PEDS》1993,6(7):691-700
A 3-D model of the human 5-HT1a receptor was constructed fromits amino acid sequence by computer graphics techniques, molecularmechanics calculations and molecular dynamics simulations. Themodel has seven -helical membrane spanning segments, which forma central core containing a putative ligand binding site. Electrostaticpotentials 1.4 Å outside the water accessible surfacewere mainly negative on the synaptic side of the receptor modeland at the postulated ligand binding site, and positive in thecytoplasmic domains. The negative electrostatic potentials aroundthe synaptic domains indicate that positively charged ligandsare attracted to the receptor by electrostatic forces. Moleculardynamics simulations of the receptor model with serotonin, ipsapirone,R()-methiothepin or S(+)- methiothepin in the centralcore suggested that up to 22 different amino acid residues mayform a ligand binding pocket, and contribute to the specificityof ligand recognition and binding. 相似文献
12.
Sylte Ingebrigt; Edvardsen Oyvind; Dahl Svein G. 《Protein engineering, design & selection : PEDS》1996,9(2):149-160
A three-dimensional model of the human 5-HT1a receptor was constructedby molecular modelling, and the molecular and electronic structuresof (R)- and (S)-5-fluoro-8-hydroxy-2-(dipropylamino)tetralin(UH-301) and of (R)- and (S)-8-hydroxy-2-(dipropylamino)tetralin(8-OH-DPAT) were examined by molecular mechanics and quantummechanics calculations and molecular dynamics simulations. Thereceptor model has seven transmembrane helices (TMHs), organizedaccording to a projection map of visual rhodopsin, and includesall loops between helices and the N- and C-terminal parts. Interactionsof UH-301 and 8-OH-DPAT with the 5-HT1a receptor were examinedby molecular dynamics simulations and energy minimization ofreceptorligand complexes. 8-OH-DPAT had lower electrostaticpotentials around the hydroxyl group and stronger hydrogen bondingto the receptor model than had UH-301. The simulations indicatedthat the 5-HT1a receptor agonists, (R)- and (S)-8-OH-DPAT and(R)-UH-301, interacted with the receptor at a site closer toAsp82 in TMH2 than did (S)-UH-301, which is a 5-HT,1a receptorantagonist. Simulations of receptorligand complexes indicatedthat Asp82, Asp116, Ser199, Thr200 and De385 are essential forbinding of both agonist and antagonist to the receptor. 相似文献
13.
Peters I.D.; Hew C.L.; Davies P.L. 《Protein engineering, design & selection : PEDS》1989,3(2):145-151
A semisynthetic winter flounder antifreeze proprotein (proAFP)coding region was constructed and inserted into a lacZ expressionvector. ProAFP was produced from the vector in Escherichia colias a C-terminal fusion to the first 289 amino acids of ß-galactosidase(ß-gal). The proAFP and ß-gal domains ofthe ß-galproAFP fusion protein were separatedby the recognition signal for the blood coagulation protease,factor Xa. Upon induction with isopropylthio-ß-D-galactosidethe fusion protein accumulated to levels of 15% of the totalprotein. The ß-galproAFP fusion protein waspartially purified by differential centrifugation, but requiredsolubilization prior to factor Xa digestion. The solubilizedfusion protein was efficiently and correctly cleaved by factorXa, after which the proAFP was purified by gel permeation. BacterialproAFP was indistinguishable from natural proAFP by the criteriaof antifreeze activity, amino-terminal sequence (15 cycles),reverse-phase HPLC and SDSpolyacrylamide gel electrophoresis.Circular dichroism measurements showed that proAFP is a compositeof random coil and -helical secondary structure, with an -helicalcontent of 44% at 0°C. It seems probable that the C-terminalregion of proAFP, which corresponds to the mature AFP protein,is mainly -helical, and that the N-terminal pro-segment is randomcoiled. 相似文献
14.
Gustchina Alia; Sansom Clare; Prevost Martine; Richelle Jean; Wodak Shoshana Y.; Wlodawer Alexander; Weber Irene T. 《Protein engineering, design & selection : PEDS》1994,7(3):309-316
The interactions between HIV1 protease and its boundinhibitors have been investigated by molecular mechanics calculationsand by analysis of crystal structures of the complexes in orderto determine general rules for inhibitor and substrate bindingto the protease. Fifteen crystal structures of HTV1 proteasewith different peptidomimetic inhibitors showed conservationof hydrogen bond interactions between the main chain C=O andNH groups of the inhibitors and the C=O and NH groups of theprotease extending from P3 C=O to P3' NH. The mean length ofthe hydrogen bonds between the inhibitor and the flexible flapsand the conserved water molecule (2.9 À) is slightlyshorter than the mean length of hydrogen bonds between the inhibitorand the more rigid active site region (3.1 À) of theprotease. The two hydrogen bonds between the conserved waterand P2 and P1' carbonyl oxygen atoms of the inhibitor are theshortest and are predicted to be important for the tight bindingof inhibitors. Molecular mechanics analysis of three crystalstructures of HIV-1 protease with different inhibitors withindependent calculations using the programs Discover and Brugelgave an estimate of 56-68% for the contribution of all the inhibitormain chain atoms to the total calculated proteaseinhibitorinteraction energy. The contribution of individual inhibitorresidues to the interaction energy wascalculated using Brugel.The main chain atoms of residue P2 had a consistently largefavorable contribution to the total interaction energy, probablydue to the presence of the two short hydrogen bonds to the flexibleflap. The contribution of individual inhibitor side chains dependedon the size of the side chain and the presence of specific hydrogenbond interactions with the protease. 相似文献
15.
Erwin C.R.; Barnett B.L.; Oliver J.D.; Sullivan J.F. 《Protein engineering, design & selection : PEDS》1990,4(1):87-97
Variants designed using PROTEUS have been produced in an attemptto engineer stabilizing salt bridges into subtilisin BPN'. Allthe mutants constructed by site-directed mutagenesis were secretedby Bacillus subtillus, except L75K. Q19E, expressed as a singlevariant and also in a double variant, Q19E/Q271E, appears toform a stabilizing salt bridge based on X-ray crystal structuredetermination and differential scanning calorimeter measurements.Although the double mutant was found to be less thermodynamicallystable than the wild-type, it did exhibit an autolytic stabilityabout two fold greater under hydrophobic conditions. Four variants,A98K, S89E, V26R and L235R, were found to be nearly identicalto wild-type in thermal stability, indicative of stable structureswithout evidence of salt bridge formation. Variants Q271E, V51Kand T164R led to structures that resulted in varying degreesof thermodynamic and autolytic instability. A computer-modelinganalysis of the PROTEUS predictions reveals that the low percentageof salt bridge formation is probably due to an overly simplisticelectrostatic model, which does not account for the geometryof the pairwise interactions. 相似文献
16.
Thunnissen M.G.M. Marjolein; Franken Peet A.; de Haas Gerard H.; Drenth Jan; Kalk Kor H.; Verheij Hubertus M.; Dijkstra W. 《Protein engineering, design & selection : PEDS》1992,5(7):597-603
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 6266 had been deleted (6266PLA2).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 6266PLA2, 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. 相似文献
17.
Sequence and structure of VH domain from naturally occurring camel heavy chain immunoglobulins lacking light chains 总被引:9,自引:0,他引:9
Muyldermans S.; Atarhouch T.; Saldanha J.; Barbosa J.A.R.G.; Hamers R. 《Protein engineering, design & selection : PEDS》1994,7(9):1129-1135
We cloned 17 different PCR fragments encoding VH genes of camel(Camelus dromedarius). These clones were derived from the camelheavy chain immunoglobulins lacking the light chain counterpartof normal immunoglobulins. Insight into the camel VH sequencesand structure may help the development of single domain antibodies.The most remarkable difference in the camel VH, consistent withthe absence of the VL interaction, is the substitution of theconserved Leu45 by an Arg or Cys. Another noteworthy substitutionis the Leu11 to Ser. This amino acid normally interacts withthe CH1 domain, a domain missing in the camel heavy chain immunoglobulins.The nature of these substitutions agrees with the increasedsolubility behavior of an isolated camel VH domain. The VH domainsof the camels are also characterized by a long CDR3, possiblycompensating for the absence of the VL contacts with the antigen.The CDR3 lacks the salt bridge between Arg94 and Asp101. However,the frequent occurrence of additional Cys residues in both theCDR1 and CDR3 might lead to the formation of a second internaldisulfide bridge, thereby stabilizing the CDR structure as inthe DAW antibody. Within CDRs of the camel VH domains we observea broad size distribution and a different amino acid patterncompared with the mouse or human VH. Therefore the camel hypervariableregions might adopt structures which differ substantially fromthe known canonical structures, thereby increasing the repertoireof the camel antigen binding sites within a VH. 相似文献
18.
Evolutionarily conserved hydrophobic residues at the core of protein structures are generally assumed to play a structural role in protein folding and stability. Recent studies have implicated that their importance to protein structures is uneven, with a few of them being crucial and the rest of them being secondary. In this work, we explored the possibility of employing this feature of native structures for discriminating non-native structures from native ones. First, we developed a network tool to quantitatively measure the structural contributions of individual amino acid residues. We systematically applied this method to diverse fold-type sets of native proteins. It was confirmed that this method could grasp the essential structural features of native proteins. Next, we applied it to a number of decoy sets of proteins. The results indicate that such an approach indeed identified non-native structures in most test cases. This finding should be of help for the investigation of the fundamental problem of protein structure prediction. 相似文献
19.
Fujiyoshi-Yoneda T.; Yoneda S.; Kitamura K.; Amisaki T.; Ikeda K.; Inoue M.; Ishida T. 《Protein engineering, design & selection : PEDS》1991,4(4):443-450
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. 相似文献
20.
Modeling of transmembrane seven helix bundles 总被引:4,自引:0,他引:4
Cronet Philippe; Sander Chris; Vriend Gert 《Protein engineering, design & selection : PEDS》1993,6(1):59-64
Transmembrane seven helix bundles form a large family of membraneinserted receptors and are responsible for a wide range of biologicalfunctions. Experimental data suggest that their overall structureis similar to bacteriorhodopsin. We describe here a new approachfor the modeling of transmembrane seven helix bundles basedon statistically derived environmental preference parameterscombined with experimentally determined features of the receptors.The method was used to create a model for the human ß2-adrenoreceptor.This model is physically plausible, is in reasonable agreementwith experimental data and may be helpful in planning new receptorengineering experiments. 相似文献