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1.
Display of {beta}-lactamase on the Escherichia coli surface: outer membrane phenotypes conferred by Lpp'-OmpA'-{beta}-lactamase fusions 总被引:3,自引:0,他引:3
Georgiou George; Stephens Daren L.; Stathopoulos Christos; Poetschke Heather L.; Mendenhall John; Earhart Charles F. 《Protein engineering, design & selection : PEDS》1996,9(2):239-247
Bacterial cell-surface exposure of foreign peptides and solubleproteins has been achieved recently by employing a fusion proteinmethodology. An Lpp'OmpA(46159)Bla fusionprotein has been shown previously to display the normally periplasmicenzyme ß-lactamase (Bla) on the cell surface of theGram-negative bacterium Escherichia coli. Here, we have investigatedthe role of the OmpA domain of the tripartite fusion proteinin the surface display of the passenger domain (Bla) and havecharacterized the effects of the fusion proteins on the integrityand permeability of the outer membrane. We show that in additionto OmpA(46159), a second OmpA segment, consisting ofamino acids 4666, can also mediate the display of Blaon the cell surface. Other OmpA domains of various lengths (aminoacids 4684, 46109, 46128, 46141and 46145) either anchored the Bla domain on the periplasmicface of the outer membrane or caused a major disruption of theouter membrane, allowing the penetration of antibodies intothe cell. Detergent and antibiotic sensitivity and periplasmicleakage assays showed that changes in the permeability of theouter membrane are an unavoidable consequence of displayinga large periplasmic protein on the surface of E.coli. This isthe first systematic report on the effects that cell surfaceengineering may have on the integrity and permeability propertiesof bacterial outer membranes. 相似文献
2.
Strong inhibition of fibrinogen binding to platelet receptor {alpha}IIb{beta}3 by RGD sequences installed into a presentation scaffold 总被引:5,自引:0,他引:5
Lee Grace; Chan Winnie; Hurle Mark R.; DesJarlais Renee L.; Watson Felicia; Sathe Ganesh M.; Wetzel Ronald 《Protein engineering, design & selection : PEDS》1993,6(7):745-754
In order to probe the structural constraints on binding of RGDsequences to the platelet receptor IIbß3 we have usedrecombinant DNA techniques to install the RGD sequence intopresentation scaffolds, small proteins of known3-D structure chosen to present guest sequences in constrainedorientations. Using Escherichia coli expression systems we madesequence variants in which loop residues of the immunoglobulinVL domain REI and of human interleukin-1ß were replaced(without changing polypeptide length) by the RGD sequence atpositions predicted, based on small molecule studies, to orientthe RGD moiety into an active conformation. These variants donot compete for fibrinogen binding to IIbß3 up toalmost 1 mM concentration. Unfolded or proteolytically fragmentedforms of these same proteins do compete, however, showing thatthe RGD sequences in the mutants must be prohibited from bindingby constraints imposed by scaffold structure. To suppress theeffects of such structural constraints we constructed two sequencevariants in which RGD-containing sequences 4257 or 4455from the snake venom platelet antagonist kistrin were inserted(this increasing the length of the loop) into the third complementaritydetermining loop of REI. Both of these variants compete stronglyfor fibrinogen binding with IC50s in the nM range. These results,plus data on kistrin-related peptides also presented here, suggestthat the molecular scaffold REI is capable of providing to aninstalled sequence a structural context and conformation beneficialto binding. The results also suggest that in order to bind wellto IIbß3, RGD sequences in protein ligands must eitherproject significantly from the surface of the scaffold and/orretain a degree of conformational flexibility within the scaffold.Molecular scaffolds like REI should prove useful in the elucidationof structure-function relationships and the discovery of newactive sequences, and may also serve as the basis for noveltherapeutic agents. 相似文献
3.
By random approaches we have previously isolated many variantsof Escherichia coli ß-galactosidase within a shortcontiguous tract near the N-terminus (residues 812 ofwildtype enzyme), some of which have increased stability towardsheat and denaturants. The activity of these mutants was originallyanalysed and quantitated in situ in activity gels without theaddition of magnesium ions to the buffer system. We now showthat the improved stability is only observable under such conditionsof limiting magnesium ion concentrations or in the presenceof appropriate concentrations of a metal chelator. In the presenceof EDTA, purified preparations of one of these mutant enzymeswere much more resistant to denaturants than wild-type, butthis differential was completely nullified in the presence of1 mM Mg2+. However, the stability of this mutant enzyme in EDTAwas lower than that shown by it, or the wild-type enzyme, inthe presence of magnesium ions. In addition, certain alterationswithin another N-terminal tract (residues 2731 of wild-type)resulted in enzymes with greater dependence on Mg2+ than naturalß-galactosidase. We conclude that a small number ofresidue changes in a large protein can profoundly modulate therequirement for metal ion stabilization, allowing partial abrogationof this need in certain cases. Thus, some enzymes which requiredivalent metal ions for structural purposes only may be engineeredtowards metal independence. 相似文献
4.
Engels J.W.; Glauder J.; M?llner H.; Tripier D.; Uhlmann E.; Wetekam W. 《Protein engineering, design & selection : PEDS》1987,1(3):195-199
By chemoenzymatic synthesis the gene for a (Leu27) analogueof human growth hormone releasing hormone-Gly45 [(Leu27GHRH-Gly45]was constructed, cloned and expressed in Escherichia coli asa fusion protein with ß-galactosidase under the controlof the lac promoter and operator. Upon induction with isopropyl-D-thio-ß-galactopyranosidethe fusion protein accumulated to a yield of 1520% ofthe total cellular protein. After cyanogen bromide deavage ofthe fusion protein the precursor peptide (Leu27)hGHRH-Gly45was separated by extraction and purified by ion exchange andh.p.l.c.-RP18 chromatography. The purified peptide was analysedby sequencing, isolectric focusing, amino acid analysis andamino acid analysis after V8 protease digestion. The carboxy-terminalglydne was subsequently amidated by PAM (peptidylglycine--amidating-monooxygenase),an enzyme which was isolated and characterized from fresh bovinepituitaries. Correct amidatlon of the penultimate amino acid,leucine, was verified by peptide sequencing with an authenticleucine amide reference. 相似文献
5.
Multimer formation as a consequence of separate homodimerization domains: the human c-Jun leucine zipper is a transplantable dimerization module 总被引:1,自引:0,他引:1
Riley L.G.; Ralston G.B.; Weiss A.S. 《Protein engineering, design & selection : PEDS》1996,9(2):223-230
Human c-Jun and c-Fos leucine zipper domains were examined fortheir ability to serve as autonomous dimerization domains aspart of a heterologous protein construct. Schistosoma japonicumglutathione S-transferase (GST) was fused to recombinant Junleucine zipper (rJunLZ) and Fos leucine zipper (rFosLZ) domains.SDSPAGE snapshot analyses based on disulphidelinkage of monomers demonstrated the ability of rJunLZ to functionas a dimerization motif in a foreign protein environment. Sterichindrance prevented formation of rJunLZGST::rFosLZGSTheterodimers whereas rJunLZGST::rFosLZ and rJunLZ::rFosLZGSTformed readily. Furthermore, rJunLZGST generated homodimerssuggesting fusion protein heterodimers interact differentlyto homodimers. Gel filtration chromatography confirmed thatGST is a dimer in solution and that attachment of a leucinezipper domain allows further interactions to take place. Sedimentationequilibrium analyses showed that GST is a stable dimer (Ka >106 M-1) with no higher multimeric forms. rFosLZGST weaklyassociates beyond a dimer (Ka {small tilde}4x105 M-1) and rJunLZGSTassociates indefinitely (Ka {small tilde}4x106 M-1), consistentwith an isodesmic model of association. The interaction of theseleucine zippers independently of GST association demonstratestheir utility in the modification of proteins when multimerformation is desired. 相似文献
6.
Simons Guus; van den Heuvel Wim; Reynen Theo; Frijters Adri; Rutten Ger; Slangen Charles J.; Groenen Martien; de Vos Willem M.; Siezen Roland J. 《Protein engineering, design & selection : PEDS》1993,6(7):763-770
A cDNA clone containing the entire coding region for bovineß-casein A3 flanked by 53 base pairs of 5' non-codingand 358 base pairs of 3' non-coding sequences was isolated froma bovine mammary cDNA phagemid library. The coding segment formature ß-casein was subcloned into the T7 expressionsystem, in which the expression of recombinant ß-caseinwas controlled by the T7 gene 10 promoter and ribosome bindingsite. High level expression of Met-ß-casein to 20%of the total soluble proteins was obtained in Escherichia coliwithin 2 h after induction of T7 RNA-polymerase synthesis. Inan attempt to induce secretion the coding segment for matureß-casein was coupled to the ompA translations initiationsignal and signal peptide coding sequence but no secretion ofthe fusion protein and no processing of the signal peptide fromthe fusion protein was observed. Instead, the Met-ß-caseincould be isolated in asoluble form from E.coli cells after anosmotic shock, indicative of a periplasmic location. This proceduredid not lyse the cells. The protein was purified to homogeneityafter a pH 4.8 isoelectric precipitation followed by reversed-phasehigh-performance liquid chromatography. The ß-caseincDNA was altered to change the main chymosin cleavage siteinß-casein at position 192193 in two ways, namelyfrom LeuTyr to ProPro and to Leustop. Thesemutations were designed to prevent generation of the bitterpeptide ßcasein(193209) by chymosin cleavage.The mutant Met-ß-caseins were expressed in E.colito the same level as wild-type Met-ß-casein. Purifiedmutant Met-ß-casein(Prol92 Prol93) was no longerhydrolysed by chymosin at the 192193 bond. 相似文献
7.
Moser Rudolf; Frey Stefan; Munger Karl; Hehlgans Thomas; Klauser Stephan; Langen Hanno; Winnacker Ernst-L; Mertz Ronald; Gutte Bernd 《Protein engineering, design & selection : PEDS》1987,1(4):339-343
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
8.
Zinn-Justin Sophie; Pillet Laurence; Ducancel Frdric; Thomas Aline; Smith Jeremy C.; Boulain Jean-Claude; Mnez Andr 《Protein engineering, design & selection : PEDS》1994,7(7):917-923
Structural features associated with the ability of a monoclonalantibody (mAb) to discriminate between protein variants areidentified and engineered. The variants are the curaremimetictoxin from Naja nigricollis and erabutoxin a or b from Laticaudasemifasciata which differ from each other by 16 substitutionsand one insertion. The neutralizing mAb M1 recognizes with highaffinity a topographical epitope on the surface of toxin , butfails to recognize the erabutoxins although they possess mostof the residues forming the presumed epitope. Examinations ofthe toxin and erabutoxin 3-D structures and molecular dynamicssimulations reveal several differences between the variants.In particular, the region involving the ß-turn 1724is organized differently. Analysis of the differences foundin this region suggests that the insertion (or deletion) atposition 18 of the variant amino add sequences is particularlyimportant in determining the differential cross-reactivity.To test this proposal, residue 18 was deleted in one erabutoxinusing sitedirected mutagenesis, and the biological propertiesof the resulting mutant were examined. We found that full antigenicitywas restored in the previously unrecognized variant. The implicationsof this finding are discussed. 相似文献
9.
Chiche L.; Heitz A.; Padilla A.; Le-Nguyen D.; Castro B. 《Protein engineering, design & selection : PEDS》1993,6(7):675-682
The trypsin carboxypeptidase peptide inhibitor (TCPI) whichinhibits both trypsin and carboxypeptidase A has been chemicallyengineered by modification of the Ecballium elaterium trypsininhibitor II (EETI-II). The solution conformation of TCPI, studiedby two-dimensional nuclear magnetic resonance, was shown tobe very close to those of squash inhibitors. Only limited deviationsof the trypsin binding loop compared to its location in theEETI-II/trypsin complex were detected. It was also shown thatthe position of the C-terminal tail did not significantly changefrom the position observed in the complex between carboxypeptidaseA and the potato carboxypeptidase inhibitor (PCI). The conformationof TCPI was carefully compared with the PCI one and a new structuralalignment between the two microproteins is proposed. This alignmentpoints out the very good conservation in the two inhibitorsof a subdomain comprising segments 715, 1922 and2528. Most importantly, the 219 disulfide bridgeof TCPI was not structurally conserved in PCI and appeared tobe rather unimportant for the early folding process of thesemolecules. This result agrees with the recent observation thatthe 219 bridge is the last to be formed in the foldingof the squash inhibitor EETI-II and suggests that this is alsothe case during the folding of the potato carboxypeptidase inhibitor. 相似文献
10.
Sowdhamini R.; Srinivasan N.; Shoichet Brian; Santi Daniel V.; Ramakrishnan C.; Balaram P. 《Protein engineering, design & selection : PEDS》1989,3(2):95-103
A computer modeling procedure for assessing the stereochemicalsuitability of pairs of residues in proteins as potential sitesfor introduction of cystine disulfide crosslinks has been developed.Residue pairs with C C distances of 6.5 Å andCbeta;Cß distances of 4.5 Å are chosenfor geometrical fixation of S atoms using the program MODIP.The stereochemistry of the modeled disulfides is evaluated usinglimits for the structural parameters of the various torsionangles and SS bond length in the disulfide bridge. Theability of the procedure to correctly model disulfides has beenchecked with examples of cystine peptides of known crystal structuresand 103 disulfide bridges from 25 available protein crystalstructures determined at 2 Å resolution. An analysis ofresults on three proteins with engineered disulfides, T4 lysozyme,dihydrofolate reductase and subtilisin, is presented. Two positionsfor the introduction of stereochemically optimaldisulfides are identified in subtilisin. 相似文献
11.
Contreras Carlos F.; Canales Mauricio A.; Alvarez Alejandra; de Ferrari Giancarlo V.; Inestrosa Nibaldo C. 《Protein engineering, design & selection : PEDS》1999,12(11):959-966
The main component of the amyloid senile plaques found in Alzheimer'sbrain is the amyloid-ß-peptide (Aß), a proteolyticproduct of a membrane precursor protein. Previous structuralstudies have found different conformations for the Aßpeptide depending on the solvent and pH used. In general, theyhave suggested an -helix conformation at the N-terminal domainand a ß-sheet conformation for the C-terminal domain.The structure of the complete Aß peptide (residues 140)solved by NMR has revealed that only helical structure is presentin Aß. However, this result cannot explain the large ß-sheetAß aggregates known to form amyloid under physiologicalconditions. Therefore, we investigated the structure of Aßby molecular modeling based on extensive homology using theSmith and Waterman algorithm implemented in the MPsrch program(Blitz server). The results showed a mean value of 23% identitywith selected sequences. Since these values do not allow a clearhomology to be established with a reference structure in orderto perform molecular modeling studies, we searched for detailedhomology. A 28% identity with an /ß segment of a triosephosphateisomerase (TIM) from Culex tarralis with an unsolved three-dimensionalstructure was obtained. Then, multiple sequence alignment wasperformed considering Aß, TIM from C.tarralis and anotherfive TIM sequences with known three-dimensional structures.We found a TIM segment with secondary structure elements inagreement with previous experimental data for Aß. Moreover,when a synthetic peptide from this TIM segment was studied invitro, it was able to aggregate and to form amyloid fibrils,as established by Congo red binding and electron microscopy.The Aß model obtained was optimized by molecular dynamicsconsidering ionizable side chains in order to simulate Aßin a neutral pH environment. We report here the structural implicationsof this study. 相似文献
12.
The parallel ß-barrel is a recurrent structural motiffound in a large variety of different enzymes belonging to thefamily of /ß-proteins. It has been shown previouslythat the hyperboloid can be considered as a scaffold describingthe parallel ß-barrel structure. To assess restraintson ß-strand twist imposed by a given scaffold geometry,the notion of scaffold twist, Ts, is introduced. From Ts, theß-strand twist (Twß) expected for a givenscaffold geometry can be derived and it is verified that thiscomputed twist can be used to identify ß-barrels characterizedby good hydrogen bonding. It is noted that Twß isonly slightly affected for ß-barrels differing inthe number (N) of ß-strands, suggesting that restraintson main-chain conformation of ß-strands are not likelyto account for the N = 8 invariability observed in natural parallelß-barrels thereby strengthening previous work rationalizingthis constancy. 相似文献
13.
Boeggeman Elizabeth E; Balaji Petety V; Sethi Navin; Masibay Arni S; Qasba Pradman K 《Protein engineering, design & selection : PEDS》1993,6(7):779-785
Bovine ß-1, 4-galactosyltransferase (ß-1,4-GT; EC 2.4.1.90
[EC]
) belongs to the glycosyltransferase familyand as such shares a general topology: an N-terminal cytoplasmictail, a signal anchor followed by a stem region and a catalyticdomain at the C-tenninal end of the protein. cDNA constructsof the N-terminal deleted forms of ß-1, 4-GT wereprepared in pGEX-2T vector and expressed in E.coli as glutathione-S-transferase(GST) fusion proteins. Recombinant proteins accumulated withininclusion bodies as insoluble aggregates that were solubilizedin 5 M guanidine HCl and required an oxido-shufflingreagent for regeneration of the enzyme activity. The recombinant(ß-1, 4-GT, devoid of the GST domain, has 3085%of the sp. act. of bovine milk ß-1, 4-GT with apparentKms for N-acetylglucosamine and UDP-galactose similar to thoseof milk enzyme. Deletion analysesshow that both (ß-1,4-GT and lactose synthetase activities remain intact even inthe absence of the first 129 residues (pGT-dl29). The activitiesare lost when either deletions extend up to residue 142 (pGT-dl42)or Cysl34 is mutatedto Ser (pGT-dl29C134S). These results suggestthat the formation of a disulfide bond involving Cysl34 holdsthe protein in a conformation that is required for enzymaticactivity. 相似文献
14.
Imoto Taiji; Yamada Hidenori; Yasukochi Takanori; Yamada Eiichi; Ito Yuji; Ueda Tadashi; Nagatani Hiroko; Miki Takeyoshi; Horiuchi Tadao 《Protein engineering, design & selection : PEDS》1987,1(4):333-338
In the preceding paper in this issue, we described the overproduction of one mutant chicken lysozyme in Escherichia coil.Since this lysozyme contained two amino acid substitutions (Ala31ValandAsn106Ser)in addition to an extra methionine residue at theNH2-terminus the substituted amino acid residues were convertedback to the original ones by means of oligonucleotide-directedsite-specific mutagenesis and in vitro recombination. Thus fourkinds of chicken lysozyme [Met1 Val31Ser106-, Met1Ser106-,Met1 Val31-and Met1 (wild type)] wereexpressed in E. coli. From the results of folding experimentsof the reduced lysozymes by sulfhydryl-disulfide interchangeat pH 8.0 and 38°C, follow ed by the specific activity measurementsof the folded en zymes, the following conclusions can be drawn:(i) an extra methionine residue at the NH2-terminus reducesthe folding rate but does not affect the lysozyme activity ofthe folded enzyme; (ii) the substitution of Asn106 by Ser decreasesthe activity to 58% of that of intact native lysozyme withoutchanging the folding rate; and (iii) the substitution of Ala31Val prohibits the correct folding of lysozyme. Since the wildtype enzyme (Met1-lysozyme) was activated in vitro withoutloss of specific activity, the systems described in this study(mutagenesis, overproduction, purification and folding of inactivemutant lysozymes) may be useful in the study of folding pathways,expression of biological activity and stability of lysozyme. 相似文献
15.
Cols Neus; Romero-Isart Nuria; Bofill Roger; Capdevila Merce; Gonzalez-Duarte Pilar; Gonzalez-Duarte Roser; Atrian Silvia 《Protein engineering, design & selection : PEDS》1999,12(3):265-269
The ß domain of mouse metallothionein 1 (ßMT) wassynthesized in Escherichia coli cells grown in the presenceof copper or cadmium. Homogenous preparations of CußMTand CdßMT were used to characterize the correspondingin vivo-conformed metal-clusters, and to compare them with thespecies obtained in vitro by metal replacement to a canonicalZn3ßMT structure. The copper-containing ßMTclusters formed inside the cells were very stable. In contrast,the nascent ß peptide, although it showed cadmium bindingability, produced a highly unstable species, whose stoichiometrydepended upon culture conditions. The absence of ßMT proteinin E.coli protease-proficient hosts grown in cadmium-supplementedmedium pointed to drastic proteolysis of a poorly folded ßpeptide, somehow enhanced by the presence of cadmium. Possiblefunctional and evolutionary implications of the bioactivityof mammalian ßMT in the presence of monovalent and divalentmetal ions are discussed. 相似文献
16.
Jimenez M.A.; Arnold J.R.P.; Andrews J.; Thomas J.A.; Roberts G.C.K.; Birdsall B.; Feeney J. 《Protein engineering, design & selection : PEDS》1989,2(8):627-631
The complex of Lactobacillus casei dihydrofolate reductase withthe substrate folate and the coenzyme NADP* has been shown toexist in solution as a mixture of three slowly interconvertingconformations whose proportions are pH-dependent and which differin the orientation of the pteridine ring of the substrate inthe binding site. The Asp26 Asn mutant of L. casei dihydrofolatereductase has been prepared by oligonucleotide-directed mutagenesisand studied by one-and two-dimensional 1H-NMR spectroscopy.NMR studies of the mutant enzymefolateNADP* complexshow that this exists to > 90% in a single conformation overthe pH* range 57.1. The single conformation observedcorresponds to conformation I (the methotrexate-likeconformation) of the wild-type enzymefolateNADP*complex. These observations demonstrate that Asp26 is the ionizablegroup controlling the pH-dependence of the conformational equilibriumseen in the wild-type enzyme. 相似文献
17.
Pons Jaume; Planas Antoni; Querol Enrique 《Protein engineering, design & selection : PEDS》1995,8(9):939-945
Bacillus 1,3-1,4-ß-glucanases possess a highly conserveddisulfide bridge connecting a ß-strand with a solventexposedloop lying on top of the extended binding site cleft The contributionof the disulfide bond and of both individual cysteines (Cys61and Cys90) in the Bacillus licheniformis enzyme to stabilityand activity has been evaluated by protein engineering methods.Reduction of the disulfide bond has no effect on kinetic parameters,has only a minor effect on the activity-temperature profileat high temperatures, and destabilizes the protein by less than0.7 kcal/mol as measured by equilibrium urea denatu ration at37°C. Replacing either of the Cys residues with Ala destabilizesthe protein and lowers the specific activity. C90A retains 70%of wild-type (wt) activity (in terms of Vmax), whereas C61Aand the double mutant C61AC90A have 10% of wt Vmax. Alarger change in free energy of unfolding is seen by equilibriumurea denaturation for the C61A mutation (loop residue, 3.2 kcal/molrelative to reduced wt) as compared with the C90A mutation (ß-strandresidue, 1.8 kcal/mol relative to reduced wt), while the doublemutant C61AC90A is 0.8 kcal/mol less stable than thesingle C61A mutant. The effects on stability are interpretedas a result of the change in hydrophobic packing that occursupon removal of the sulfur atoms in the Cys to Ala mutations 相似文献
18.
Sequence variants of the ß-barrel protein interleukin-1ßhave been analyzed for their stabilities toward irreversiblethermal inactivation by monitoring the generation of light scatteringaggregates on heating. The derived temperatures for the onsetof aggregation (Tagg values) correlate well with the free energiesof unfolding of these proteins with the exception of one variant,Lys97Val (K97V), which undergoes aggregation at a temperature7°C lower than expected based on its thermodynamic stability.This lower than expected thermal stability may be due to generationof an aggregation-prone unfolding intermediate at a temperaturelower than the Tm of the global transition. This hypothesisis supported by the location of residue 97 in the long 8699loop which has structural features suggesting it may comprisea small, independent folding unit or microdomain. The excellentcorrelation of thermal and thermodynamic stabilities of sevenof the eight variants tested is consistent with accepted modelsfor thermal inactivation of proteins. At the same time the poorfit of the K97V variant underscores the risk in using thermalstability data in quantitative analysis of mutational studiesof the folding stability of proteins. 相似文献
19.
Delagrave Simon; Goldman Ellen R.; Youvan Douglas C. 《Protein engineering, design & selection : PEDS》1995,8(3):237-242
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
20.
Site-directed and deletion mutational analysis of the receptor binding domain of the interleukin-6 receptor targeted fusion toxin DAB389-IL-6 总被引:1,自引:0,他引:1
Jean Lee-Fong L.; Waters Cory A.; Keemy Donald; Murphy John R. 《Protein engineering, design & selection : PEDS》1993,6(3):305-311
We have used site-directed and in-frame deletion mutationalanalysis in order to explore the structural features of theIL6 portion of the diphtheria toxin-related interleukin6(IL6) fusion toxin DAB389-IL6 that are essentialfor receptorbinding and subsequent inhibition of protein synthesisin target cells. Deletion of the first 14 amino acids of theIL6 component of the fusion toxin did not alter eitherreceptor binding affinity or cytotoxk potency. In contrast,both receptor binding and cytotoxic activity were abolishedwhen the Cterminal 30 amino acids of the fusion toxinwere deleted. In addition, we explored the relative role ofthe disulfide bridges within the IL6 portion of DAB389-IL6in the stabilization of structure required for receptor-binding.The analysis of mutants in which the substitution of eitherCys440, Cys446, Cys469 or Cys479 to Ser respectively, demonstratesthat only the disulfide bridge between Cys469 and Cys479 isrequired to maintain a functional receptor binding domain. Inaddition, the internal in-frame deletion of residues 435451,which includes Cys440 and Cys446, was found to reduce, but notabolish receptor binding affinity. These results further demonstratethat the disulfide bridge between Cys440 and Cys446 is not essentialfor receptor-binding. However, the reduced cytotoxic potencyof DAB389-IL6(435451) suggests that the conformationand/or receptor binding sites associated with this region ofthe fusion toxin is/are important for maintaining the wild typereceptor binding affinity and cytotoxic potency. 相似文献