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1.
The crystallographic study of chimeric B72.3 antibody illustratedthat there are three FR side-chain interactions with eitherCDR residue's side chain or main chain. For example, hydrogenbonds are formed between the hydroxyl group of threonine atL5 in FR1 and the guanidinal nitrogen group of arginine at L24in CDR1, between the hydroxyl group of tyrosine at L36 in FR2and the amide nitrogen group of glutamine at L89 in CDR3 andbetween the hydroxyl group of tyrosine at L71 in FR3 and thecarbonyl group of isoleucine at L29 as well as the amide nitrogengroup of serine at L31 in CDR1. Elimination of these hydrogenbonds at these FR positions may affect CDR loop conformations.To confirm these assumptions, we altered these FR residues bysite-directed mutagenesis and determined binding affinitiesof these mutant chimeric antibodies for the TAG72 antigen. Wefound that the substitution of tyrosine by phenylalanine atL71, altering main-chain hydrogen bonds, significantly reducedthe binding affinity for the TAG72 antigen by 23-fold, whereasthe substitution of threonine and tyrosine by alanine and phenylalanineat L5 and L36, eliminating hydrogen bonds to side-chain atoms,did not affect the binding affinity for the TAG72 antigen. Ourresults indicate that the light-chain FR residue tyrosine atL71 of chimeric B72.3 antibody plays an important role in influencingthe TAG72 antigen binding. Our results will thus be of importancewhen the humanized B72.3 antibody is constructed, since thisimportant mouse FR residue tyrosine at L71 must be maintained.  相似文献   

2.
The importance of Trp H33 in antibody recognition of DNA containinga central pyrimidine (6–4) pyrimidone photoproduct wasinvestigated. This residue was replaced by Tyr, Phe and Alaand the binding abilities of these mutants were determined bysurface plasmon resonance and fluorescence spectroscopy. Conservativesubstitution of Trp H33 by Tyr or Phe resulted in moderate lossesof binding affinity; however, replacement by Ala had a significantlylarger impact. The fluorescence properties of DNA containinga (6–4) photoproduct were strongly affected by the identityof the H33 residue. DNA binding by both the wild-type and theW-H33-Y mutant was accompanied by a small degree of fluorescencequenching; by contrast, binding by the W-H33-F and W-H33-A mutantsproduced large fluorescence increases. Taken together, thesevariations in binding and fluorescence properties with the identityof the H33 residue are consistent with a role in photoproductrecognition by Trp H33 in the high-affinity antibody 64M5.  相似文献   

3.
Starting from a synthetic modular gene (infA*) encoding Escherichiacoli translation initiation factor IF1, we have constructedmutants in which amino acids are deleted from the carboxyl terminusor in which His29 or His34 are replaced by Tyr or Asp residues.The mutant proteins were overproduced, purified and tested invitro for their properties in several partial reactions of thetranslation initiation pathway and for their capacity to stimulateMS2 RNA-dependent protein synthesis. The results allow for theconclusion that: (i) Arg69 is part of the 30S ribosomal subunitbinding site of IF1 and its deletion results in the substantialloss of all IF1 functions; (ii) neither one of its two histidinesis essential for the binding of IF1 to the 30S ribosomal subunit,for the stimulation of fMet-tRNA binding to 30S or 70S ribosomalparticles or for MS2 RNA-dependent protein synthesis; but (iii)His29 is involved in the 50S subunit-induced ejection of IF1from the 30S ribosomal subunit.  相似文献   

4.
The location of the first seven residues of the regulatory chainof Escherichia coli aspartate transcarbamoylase has been identifiedby X-ray crystallography to be near the binding site of theregulatory nucleotides. In order to determine the function ofthe N-terminus of the regulatory chain of aspartate transcarbamoylasein heterotropic regulation, alanine scanning mutagenesis wasused. Specifically, Thr2r, His3r, Asp4r, Asn5r, Lys6r and Leu7rwere each replaced with alanine. Analyses of these mutant enzymesindicate that none of these substitutions significantly alterthe catalytic properties of the enzyme. However, three of themutant enzymes, Asp4r Ala, Lys6r Ala and Leu7r Ala, exhibitednotable changes in their response to the regulatory nucleotides,while mutations at Thr2r, His3r and Asn5r exhibited only minorchanges in their heterotropic responses. For the Asp4r Alaenzyme, the responses to ATP and CTP were reduced 30 and 40%respectively, compared with the wild-type enzyme. For the Lys6r Ala enzyme, the response to ATP was reduced 70%, while theCTP response was reduced 50%. In the case of the Leu7r Alaenzyme, a 30 and 20% reduction in response to ATP and CTP respectively,was observed. The synergistk inhibition by UTP in the presenceof CTP for the Lys6r Ala enzyme was reduced 40% compared withthat of the wild type enzyme. For the Leu7r Ala enzyme, thesynergistic inhibition was abolished. In addition, UTP decreasedthe CTP binding affinity of the Leu7r Ala enzyme. Analysisof the kinetic data from these mutant enzymes suggests thatresidues Thr2r, His3r and Asn5r have little effect on the heterotropicmechanism, while residues Asp4r, Lys6r and Leu7r play a moresignificant role in the heterotropic response of the enzymetoward the nucleotides. Furthermore, residue Leu7r appears tobe directly involved in the mechanism for synergistic inhibitionof aspartate transcarbamoylase. In this study alanine scanningmutagenesis has provided a rapid method of identifying thoseresidues in the N-terminal region of the regulatory chain ofaspartate transcarbamoylase important for heterotropic regulation.  相似文献   

5.
Systematic replacement of the amino acid residues in Escherichiacoli ribonuclease HI with those in the thermophilic counterparthas revealed that two mutations, His62–Pro (H62P) andLys95Gly (K95G), increased the thermostability of the protein.These single-site mutant proteins, together with the mutantproteins His62Ala (H62A), Lys95Asn (K95N) and Lys95Ala (K95A),were crystallized and their structures were determined at 1.8Å resolution. The crystal structures of these mutant proteinsreveal that only the local structure around each mutation siteis essential for the increase in thermostability. For each mutantprotein, the stabilization mechanism is considered to be asfollows: (i) H62P is stabilized because of a decrease in theentropy of the unfolded state, without a change in the nativebackbone structure; (ii) K95G is stabilized since the straincaused by the left-handed backbone structure in the typical3:5 type loop is eliminated; and (iii) K95N is slightly stabilizedby a hydrogen bond formed between the side-chain N-atom of themutated aspargine residue and the main-chain carbonyl oxygenwithin the same residue.  相似文献   

6.
Three mutants of Escherichia coli ribonuclease HI, in whichan invariant acidic residue Asp134 was replaced, were crystallized,and their three-dimensional structures were determined by X-raycrystallography. The D134A mutant is completely inactive, whereasthe other two mutants, D134H and D134N, retain 59 and 90% activitiesrelative to the wild-type, respectively. The overall structuresof these three mutant proteins are identical with that of thewild-type enzyme, except for local conformational changes ofthe flexible loops. The ribonuclease H family has a common activesite, which is composed of four invariant acidic residues (Asp10,G1u48, Asp70 and Asp134 in E.coli ribonuclease HI), and theirrelative positions in the mutants, even including the side-chainatoms, are almost the same as those in the wild-type. The positionsof the -polar atoms at residue 134 in the wild-type, as wellas D134H and D134N, coincide well with each other. They arelocated near the imidazole side chain of His124, which is assumedto participate in the catalytic reaction, in addition to thefour invariant acidic residues. Combined with the pH profilesof the enzymatic activities of the two other mutants, H124Aand H124A/D134N, the crystallographic results allow us to proposea new catalytic mechanism of ribonuclease H, which includesthe roles for Asp134 and His124.  相似文献   

7.
A highly conserved lysine at position 128 of Escherichia coliglutamate dehydrogenase (GDH) has been altered by sitedirectedmutagenesis of the gdhA gene. Chemical modification studieshave previously shown the importance of this residue for catalyticactivity. We report the properties of mutants in which lysine-128has been changed to histidine (K128H) or arginine (K128R). Bothmutants have substantially reduced catalytic centre activitiesand raised pH optima for activity. K128H also has increasedrelative activity with amino acid substrates other than glutamate,especially L-norvaline. These differences, together with alterationsin Km values, Kd values for NADPH and K1 values for D-glutamate,imply that lysine-128 is intimately involved in either director indirect interactions with all the substrates and also incatalysis. These multiple interactions of lysine-128 explainthe diverse effects of chemical modifications of the correspondinglysine in homologous GDHs. In contrast, lysine-27, another highlyreactive residue in bovine GDH, is not conserved in all of thesequenced NADP-specific GDHs and is therefore not likely tobe involved in catalysis.  相似文献   

8.
A mutant of papain, where an inter-domain hydrogen bond betweenthe side chain hydroxyl group of a serine residue at position176 and the side chain carbonyl oxygen of a glutamine residueat position 19 has been removed by site-directed mutagenesis,has been produced and characterized kinetically. The mutationof Ser176 to an alanine has only a small effect on the kineticparameters, the kcat/Km for hydrolysis of CBZ-Phe-Arg-MCA bythe Serl76Ala enzyme being of 8.1 x 104 /M/s compared with 1.2x 105 /M/s for papain. Serine 176 is therefore not essentialfor the catalytic functioning of papain, even though this residueis conserved in all cysteine proteases sequenced. The pH-activityprofiles were shown to be narrower in the mutant enzyme by upto 1 pH unit at high ionic strength. This result is interpretedto indicate that replacing Ser 176 by an alanine destabilizesthe thiolate—imidazolium form of the catalytic site Cys25-Hisl59residues of papain. Possible explanations for that effect aregiven and the role of a serine residue at position 176 in papainis discussed.  相似文献   

9.
Lysl8, Arg86, Asn283, Ser286, Thr288 and Glu292 of glutathionesynthetase from Escherichia coli B are presumed to be highlyconcerned with the substrate, -L-glutamyl-L-cysteine (-Glu-Cys),binding by X-ray crystallography and affinity labeling studies.Using site-directed mutagenesis, we investigated functionalroles of those residues for -Glu-Cys binding. The mutant enzymesof Arg86 and Asn283 altered their kinetic parameters, especiallythe Michaelis constants of -Glu-Cys. In the case of Asn283,the residue is not likely to have an essential role in -Glu-Cysbinding but its side chain would extend to make a van der Waalscontact with bound -Glu-Cys. Chemical modification of a cysteineresidue with 5,5'-dithiobis(2-nitrobenzoate) (DTNB) showed Arg86would not only be much responsible for -Glu-Cys binding butwould also have a role in maintaining the structural integrityof the enzyme. The other mutant enzymes showed little defectin their kinetic parameters of -Glu-Cys.  相似文献   

10.
Site-specific mutagenesis has been used to probe amino acidresidues proposed to be critical in catalysis by Escherichiacoli asparaginase II. Thr12 is conserved in all known asparaginases.The catalytic constant of a T12A mutant towards L-aspartk acidß-hydroxamate was reduced to 0.04% of wild type activity,while its An, and stability against urea denaturation were unchanged.The mutant enzyme T12S exhibited almost normal activity butaltered substrate specificity. Replacement of Thr119 with Alaled to a 90% decrease of activity without markedly affectingsubstrate binding. The mutant enzyme S122A showed normal catalyticfunction but impaired stability in urea solutions. These dataindicate that the hydroxyl group of Thr12 is directly involvedin catalysis, probably by favorably interacting with a transitionstate or intermediate. By contrast, Thr119 and Ser122, bothputative target sites of the inactivator DONV, are functionallyless important.  相似文献   

11.
Cassette mutagenesis was used to exchange the suggested copperligand Met121 in azurin to all other amino acids, and a stopcodon. The mutant proteins were characterized by optical absorptionspectroscopy and EPR. At low pH, all mutants exhibit the characteristicsof a blue type 1 copper protein, indicating that methionineis not needed to create a blue copper site. At high pH, theGlu121 and the Lys121 mutants constitute a new form of protein-boundcopper that is outside the range of type1 copper.  相似文献   

12.
To understand the functional roles of Cys residues in the subunitof tryptophan synthase from Escherichia coli, single mutantsof the subunit, in which each of the three Cys residues wassubstituted with Ser, Gly, Ala or Val, were constructed by site-directedmutagenesis. The effects of the substitutions on the functionof tryptophan synthase were investigated by activity measurements,calorimetric measurements of association with the ßsubunit and steadystate kinetic analysis of catalysis. Althoughthe three Cys residues are located away from the apparentlyimportant parts for enzymatic activity, substitutions at position81 by Ser, Ala or Val caused decreases in the intrinsic activityof the subunit. Furthermore, Cys81Ser and Cys81Val reducedstimulation activities in the and ß reactions dueto formation of a complex with the ß subunit. Thelower stimulation activities of the mutant proteins were notcorrelated with their abilities to associate with the ßsubunit but were correlated with decreases in kcat. The presentresults suggest that position 81 plays an indirectly importantrole in the activity of the subunit itself and the mutual activationmechanism of the complex.  相似文献   

13.
Oligonucleotide-directed mutagenesis was employed to producemutants of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)of Escherichia coli and Bacillus stearothermophilus. Three differentmutant proteins—His176 — Asn, Cys149 — Ser,Cys149 — Gly—were isolated from one or both of theenzymes. The study of the properties of these mutants has shownthat Cys149 is clearly responsible for the information of acharge-transfer transition, named the Racker band, observedduring the NAD+ binding to apoGAPDH. This result excludes asimilarity between the Racker band and the charge-transfer transitionobserved following the alkylation of GAPDH by 3-chloroacetylpyridine-adenine dinucleotide.  相似文献   

14.
The strictly conserved active site residue, Asp222, which formsa hydrogen-bonded salt bridge with the pyridine nitrogen atomof the pyridoxal 5' phosphate (PLP) co-factor of aspartate aminotransferase(AATase), was replaced with alanine (D222A) in the Escherichiacoli enzyme. The D222A mutant exhibits non–hyberbolicsaturation behavior with amino acid substrates which appearas apparent negative eooperativity in steady–state kineticanalyses. Single turnover progress curves for D222A are welldescribed by the sum of two exponentials, contrasting with themonophasic kinetics of the wild-type enzyme. An active/inactiveheterodimer containing the D222A mutation retains this biphasickinetic response, proving that the observed eooperativity isnot the result of induced allostery. The anomalous behavioris explained by a hysteretic kinetic model involving two slowlyinterconverting enzyme forms, only one of which is catalyticallycompetent. The slow functional transition between the two formshas a half–life of 10 mins. Preincubation of the mutantwith the dicarboxylk inhibitor maleate shifts the equilibriumpopulation of the enzyme towards the catalytically active form,suggesting that the slow transition is related to the domainclosure known to occur upon association of this inhibitor withthe wild-type enzyme. The importance of Asp222 in the chemicalsteps of transamination is confirmed by the l05fold decreasein catalytic competence in the D222A mutant, and by the largeprimary C–deuterium kinetic isotope effect (6.7 versus2.2 for the wild–type). The transamination activity ofthe D222A mutant is enhanced 4– to 20–fold by reconstltutionwith the co-factor analog N–methylpyridoxal–5–phosphate(N–MePLP), and the C–proton abstraction step isless rate determining, as evidenced by the decrease in the primarykinetic isotope effect from 6.7 to 2.3. These results suggestthat the conserved interaction between the protonated pyridinenitrogen of PLP and the negatively charged carboxylate of Asp222is important not only for efficient C–proton abstraction,but also for conformational transitions concomitant with thetransamination process  相似文献   

15.
Two different combinatorial mutagenesis experiments on the light-harvestingII (LH2) protein of Rhodobacter capsulatus indicate that heuristicrules relating sequence directly to phenotype are dependenton which sets or groups of residues are mutated simultaneously.Previously reported combinatorial mutagenesis of this chromogenicprotein (based on both phylogenetic and structural models) showedthat substituting amino acids with large molar volumes at Glyß31caused the mutated protein to have a spectrum characteristicof light-harvesting I (LH1). The six residues that underwentcombinatorial mutagenesis were modeled to lie on one side ofa transmembrane -helix that binds bacteriochlorophyll. In asecond experiment described here, we have not used structuralmodels or phylogeny in choosing mutagenesis sites. Instead,a set of six contiguous residues was selected for combinatorialmutagenesis. In this latter experiment, the residue substitutedat Glyß31 was not a determining factor in whetherLH2 or LH1 spectra were obtained; therefore, we conclude thatthe heuristic rules for phenotype prediction are context dependent.While phenotype prediction is context dependent, the abilityto identify elements of primary structure causing phenotypediversity appears not to be. This strengthens the argument forperforming combinatorial mutagenesis with an arbitrary groupingof residues if structural models are unavailable.  相似文献   

16.
The acetylcholine-binding protein (AChBP) is homologous to the ligand-binding domain of the nicotinic acetylcholine receptor (nAChR) and other members of the Cys-loop family of neurotransmitter receptors. The high-resolution X-ray structures of AChBP mean it has been used as a model from which to understand agonist and antagonist binding to nAChRs. We present here a molecular dynamics (MD) study of AChBP with nicotine and carbamylcholine bound. Our results suggest that the ligand imposes rigidity on the binding pocket residues. The simulations also suggest that the protein undergoes breathing motions with respect to the five-fold axis, a motion that has been postulated to be related to gating in the nAChR. We analyzed the behaviour of the water molecules in and around the binding site and found that they occupied five distinct sites within the binding pocket. Water occupied these sites in the absence of ligand, but the presence of ligand increased the probability that a water molecule would be found in these sites. Finally, we demonstrate how the positions of these waters might be used in the design of new ligands by comparing the positions of these sites with other recent structures.  相似文献   

17.
The amino acids that are required for the cytotoxic activityof recombinant human tumor necrosis factor- (TNF) were investigatedby chemical modification and oligonucleotide-directed site-specificmutagenesis. TNF contains three histidine residues, locatedat positions 15, 73 and 78. The histidine-specific reagent diethylpyrocarbonate(DEP) was used to chemically modify TNF. The chemical inactivationof the in vitro cytotoxic activity of this lymphokine (usingmurine L929 target cells) was found to be time- and dose-dependent.Inactivated TNF failed to compete with fully bioactive [125I]TNFfor human MCF-7 target cell receptors. Mutant polypeptides ofTNF were genetically engineered by oligonucoleotide-directedsite-specific mutagenesis. The cytotoxicity of a double histidinemutant, in which histidine-73 and histidine-78 were replacedwith glutamine, was not altered and was chemically inactivatedby DEP. Substituting glutamine for histidine-15 resulted in10–15% of the wild-type bioactivity. Replacing histidine-15with either asparagine, lysine or glycine resulted in a biologicallyinactive molecule. The data show that the histidine residueat position 15 is an amino acid that is required for the cytotoxicactivity of TNF.  相似文献   

18.
Bacterial expression systems can greatly facilitate proteinengineering of antibodies. We have developed a system for high-levelexpression of antibodies, antibody fragments, or hybrid antibodieswith novel effector functions in the periplasm of Escherichiacoli. From 5 ml of cells, a simple extraction yields sufficientmaterial for SDS-gel electro-phoresis, detection and characterizationof hapten binding. To demonstrate our system, heavy-chain variableregions and 1 light chains of a mouse anti-NP antibody weresynthesized as hybrid proteins with a bacterial signal peptide(Omp F). Each chain is secreted into the periplasm where processing(cleavage of the signal peptide), folding and heterodimer associationtake place. Periplasmic proteins are released by cold osmoticshock, and hapten-binding activity is easily detected withoutfurther manipulation. The ease of genetic engineering in thissystem will facilitate the production of immunoglobulin derivativesdesigned for specific applications, and expression of thesemolecules in a native state will allow the rapid screening ofcombinatorial libraries and the results of mutagenesis.  相似文献   

19.
The stability and unfolding of an immunoglobulin (Ig) G bindingprotein based upon the B domain of protein A (SpAB) from Staphylococcusaureus were studied by substituting tryptophan residues at strategiclocations within each of the three a-helical regions (al-a3)of the domain. The role of the C-terminal helix, a3, was investigatedby generating two protein constructs, one corresponding to thecomplete SpAB, the other lacking a part of ct3; the Trp substitutionswere made in both one-and two-domain versions of each of theseconstructs. The fluorescence properties of each of the single-tryptophanmutants were studied in the native state and as a function ofguanidine-HCl-mediated unfolding, and their IgG binding activitieswere determined by a competitive enzyme-linked immunosorbentassay. The free energies of folding and of binding to IgG foreach mutant were compared with those for the native domains.The effect of each substitution upon the overall structure andupon the IgG binding interface was modelled by molecular graphicsand energy minimization. These studies indicate that (i) 3 contributesto the overall stability of the domain and to the formationof the IgG binding site in l and 2, and (ii) al unfolds first,followed by 2 and 3 together.  相似文献   

20.
This paper reports the expression of an artificial functionalpolypeptide in bacteria. The gene of a designed 24-residue DDT-bindingpolypeptide (DBP) was inserted between the BamHI and PstI cleavagesites of plasmid pUR291. The hybrid plasmid, pUR291-DBP, wascloned in Escherichia coli JM109. After induction by isopropyl-ß-D-thiogalactopyranosidea fusion protein was expressed in which DBP was linked to theCOOH-termiuus of ß-galactosidase. DBP, which is stableto trypsin, was obtained by tryptic digestion of the fusionprotein and subsequent fractionation of the tryptic peptidesby reversed-phase h.p.l.c. Recombinant and chemically synthesizedDBP showed identical chromatographic properties, amino acidcomposition, and chymotryptic digestion patterns. Both the ß-galactosidase-DBPfusion and isolated recombinant DBP bound DDT. The fusion proteinwas 25 times as potent as the designed 24-residue DBP in activatinga cytochrome P-450 model system using equimolar catalytic amountsof the two proteins.  相似文献   

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