共查询到20条相似文献,搜索用时 31 毫秒
1.
Alfthan Kaija; Takkinen Kristiina; Sizmann Dorothea; Soderlund Hans; Teeri Tuula T. 《Protein engineering, design & selection : PEDS》1995,8(7):725-731
Single-chain antibodies were constructed using six differentlinker peptides to join the VH and VL domains of an anti-2-phenyloxazolone(Ox) antibody. Four of the linker peptides originated from theinterdomain linker region of the fungal cellulase CBHI and consistedof 28, 11, six and two amino acid residues. The two other linkerpeptides used were the (GGGGS)3 linker with 15 amino acid residuesand a modified IgG2b hinge peptide with 22 residues. Proteolyticstability and Ox binding properties of the six different scFvderivatives produced in Escherichia coli were investigated andcompared with those of the corresponding Fv fragment containingno joining peptide between the V domains. The hapten bindingproperties of different antibody fragments were studied by ELISAand BIAcoreTM. The interdomain linker peptide improved the haptenbinding properties of the antibody fragment when compared withFv fragment, but slightly increased its susceptibility to proteases.Single-chain antibodies with short CBHI linkers of 11, six andtwo residues had a tendency to form multimers which led to ahigher apparent affinity. The fragments with linkers longerthan 11 residues remained monomeric. 相似文献
2.
Synthetic genes (A, AB and AHB) constructed and cloned intopKK233-2 vector were recloned from the parent plasmid into thenew procaryotic expression vectors pGFY221N and pBIO52. GeneAFB (coding for all amino acids besides phenylalanine)was obtained by cassette mutagenesis from geneAB. The plasmid pGFY221N was constructed from pGFY218L by replacingthe PstI by an NcoI site; plasmid pBIO52 was derived from pGFY221Nthrough replacing the 221-bp EoRl/NcoI fragment with a syntheticDNA segment of 52 bp representing the Escherichia coli atpEgene translational initiation region. The genes A, AB, AHB andAFB in the vector pGFY221N were expressed with a six-amino-acid-longleader sequence; in pBIO52 the genes were expressed directly.in vitro expression experiments were successful with all thegenes except with the AHB gene integrated into pGFY221N. Inthe E. coli minicell system expression was demonstrated withthe A gene in pGFY221N and the AFB and AHB genes in pBIO52.Complete translation of the expressed genes AB, AFB andAHB in either the in vitro or in vivo systems could be shownby using 35S-labelled N-terminal methionine and C-terminal cysteine.Both amino acids occur only once in the peptide sequences. 相似文献
3.
Lipid-tagged antibodies: bacterial expression and characterization of a lipoprotein--single-chain antibody fusion protein 总被引:2,自引:0,他引:2
Laukkanen Marja-Leena; Teeri Tuula T.; Keinanen Kari 《Protein engineering, design & selection : PEDS》1993,6(4):449-454
In order to achieve a stable and functional immobilization ofantibodies, we investigated the possibility of adding hydrophobicmembrane anchors to antibody fragments expressed in Escherichiacoli. The DNA sequence encoding the signal peptide and the nineN-terminal amino add residues of the major lipoprotein of E.coliwas fused to the sequence of an anti-2-phenyloxazolone single-chainFv antibody fragment [Takkinen et al. (1991) Protein Engng,4, 837841]. The expression of the fusion construct inE.coli resulted in specific accumulation of an immunoreactive28 kDa polypeptide. Unlike the unmodified single-chain Fv fragment,the fusion protein was cell-associated, labelled by [3H]palmitatewhich is indicative of the presence of N-terminal lipid modification,partitioned into the detergent phase upon Triton X-114 phaseseparation and was localized predominantly in the bacterialouter membrane. The fusion antibody displayed specific 2-phenyloxazolone-bindingactivity in the membranebound form and after solubilizationwith non-ionic detergents. Furthermore, upon removal of detergentthe fusion antibody was incorporated into proteoliposomes whichdisplayed specific hapten-binding activity. Our results showthat antibodies can be converted to membrane-bound proteinswith retention of antigen-binding properties by introductionof lipid anchors during biosynthesis. This approach may proveuseful in the design of immunoliposomes and immunosensors. 相似文献
4.
Uemura H.; Rogers M.J.; Swanson R.; Watson L.; S?ll D. 《Protein engineering, design & selection : PEDS》1988,2(4):293-296
We have used a combination of a genetic selection and oligonucleotide-directedmutagenesis to introduce a series of amino add replacementsfor a single residue into Escherichia coliglutaminyl-tRNA synthetase.The mutant enzymes mischarge supFtRNATyr, with glutamine, tovarying degrees depending on the polarity of the side chainintroduced but apparently not depending on the size or shapeof the side chain. These results indicate that repulsive charge-chargeinteractions may be important for specific recognition of nucleicacids by proteins and illustrate how a mutant, derived fromgenetic selection, may be further modified in activity by oligonucleotide-directedmutagenesis. 相似文献
5.
Delagrave Simon; Catalan John; Sweet Charles; Drabik Glenn; Henry Andrew; Rees Anthony; Monath Thomas P.; Guirakhoo Farshad 《Protein engineering, design & selection : PEDS》1999,12(4):357-362
HNK20 is a mouse monoclonal IgA that binds to the F glycoproteinof respiratory syncytial virus (RSV) and neutralizes the virus,both in vitro and in vivo. The single-chain antibody fragment(scFv) derived from HNK20 is equally active and has allowedus to assess rapidly the effect of mutations on affinity andantiviral activity. Humanization by variable domain resurfacingrequires that surface residues not normally found in a humanFv be mutated to the expected human amino acid, thereby eliminatingpotentially immunogenic sites. We describe the constructionand characterization of two humanized scFvs, hu7 and hu10, bearing7 and 10 mutations, respectively. Both molecules show unalteredbinding affinities to the RSV antigen (purified F protein) asdetermined by ELISA and surface plasmon resonance measurementsof binding kinetics (Ka 1x109 M1). A competition ELISAusing captured whole virus confirmed that the binding affinitiesof the parental scFv and also of hu7 and hu10 scFvs were identical.However, when compared with the original scFv, hu10 scFv wasshown to have significantly decreased antiviral activity bothin vitro and in a mouse model. Our observations suggest thatbinding of the scFv to the viral antigen is not sufficient forneutralization. We speculate that neutralization may involvethe inhibition or induction of conformational changes in thebound antigen, thereby interfering with the F protein-mediatedfusion of virus and cell membranes in the initial steps of infection. 相似文献
6.
Pomes R.; Willson R.C.; McCammon J.A. 《Protein engineering, design & selection : PEDS》1995,8(7):663-675
Free energy simulations are reported for the N31L-D mutation,both in the HyHEL-10-HEL antibody-lysozyme complex and in theunliganded antibody, using the thermo-dynamic-cycle perturbationmethod. The present study suggests that the mutation would changethe free energy of binding of the complex by 5.6 kcal/mol(unrestrained free energy simulations), by 0.5 kcal/mol(free energy simulations with a restrained backbone) and by1.8 kcal/ mol (Poisson-Boltzmann calculations, which also usea restrained geometry model). A detailed structural analysishelps in estimating the contributions from various residuesand regions of the system. Enhanced recognition of HEL by themutant HyHEL-10 would arise from the combination of thermodynamicallymore favorable conformational changes of the CDR loops uponassociation and subsequent charge pairing with Lys96 in theantigen. 相似文献
7.
Field Helen; Yarranton G.T.; Rees A.R. 《Protein engineering, design & selection : PEDS》1990,3(7):641-647
The expression of immunoglobulin heavy and light chain variableregions in the cytoplasm of Escherichia coli and formation ofa functional heterodimer has been demonstrated. Variable domainsequences were taken from the heavy and light chain cDNAs ofthe monoclonal antibody Gloop 2 and engineered for expressionin a dual origin expression vector. The engineered genes vhg2and vlg2 were separately subcloned into the vector, creatingtwo expression plasmids. Expression of the heavy and light chainvariable region genes (encoding 116 and 109 amino adds respectively)was investigated in eight E.coli strains; the polypeptides wererapidly degraded in a host strain optimized for expression andin E.coli strains deficient in the major protease La (lon-).Accumulation was permitted in severely protease-deficient E.colihaving a defective heat-shock response. A lon- mutation in thisgenetic background permitted even higher accumulation. Expressionlevels were 7 and 1% of total bacterial protein for light andheavy chain variable regions respectively. Expression of theheavy chain variable region gene was increased by includinga longer ShineDalgarno sequence. Similar constructionsin the light chain vector had no effect on expression levels.The insoluble variable region polypeptides were reconstitutedinto a heterodimer possessing the full antigen binding characteristicsof both the parent monoclonal antibody and its Fab fragment. 相似文献
8.
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
9.
Christmann Andreas; Walter Kerstin; Wentzel Alexander; Kratzner Ralph; Kolmar Harald 《Protein engineering, design & selection : PEDS》1999,12(9):797-806
The Ecballium elaterium trypsin inhibitor II (EETI-II), a memberof the squash family of protease inhibitors, is composed of28 amino acid residues and is a potent inhibitor of trypsin.Its compact structure is defined by a triple-stranded antiparallelß-sheet, which is held together by three intramoleculardisulfide bonds forming a cystine knot. In order to explorethe potential of the EETI-II peptide to serve as a structuralscaffold for the presentation of randomized oligopeptides, weconstructed two EETI-II derivatives, where the six-residue inhibitorloop was replaced by a 13-residue epitope of Sendai virus L-proteinand by a 17-residue epitope from human bone Gla-protein. EETI-IIand derived variants were produced via fusion to maltose bindingprotein MalE. By secretion of the fusion into the periplasmicspace, fully oxidized and correctly folded EETI-II was obtainedin high yield. EETI-II and derived variants could be presentedon the Escherichia coli outer membrane by fusion to truncatedLpp'OmpA', which comprises the first nine residues ofmature lipoprotein plus the membrane spanning ß-strandfrom residues 4666 of OmpA protein. Gene expression wasunder control of the strong and tightly regulated tetA promoter/operator.Cell viability was found to be drastically reduced by high levelexpression of Lpp'OmpA'EETI-II fusion protein.To restore cell viability, net accumulation of fusion proteinin the outer membrane was reduced to a tolerable level by introductionof an amber codon at position 9 of the lpp' sequence and utilizingan amber suppressor strain as expression host. Cells expressingEETI-II variants containing an epitope were shown to be surfacelabeled with the respective monoclonal antibody by indirectimmunofluorescence corroborating the cell surface exposure ofthe epitope sequences embedded in the EETI-II cystine knot scaffold.Cells displaying a particular epitope sequence could be enriched107-fold by combining magnetic cell sorting with fluorescence-activatedcell sorting. These results demonstrate that E.coli cell surfacedisplay of conformationally constrained peptides tethered tothe EETI-II cystine knot scaffold has the potential to becomean effective technique for the rapid isolation of small peptidemolecules from combinatorial libraries that bind with high affinityto acceptor molecules. 相似文献
10.
Gualerzi C.O.; Spurio R.; Teana A.La; Calogero R.; Celano B.; Pon C.L 《Protein engineering, design & selection : PEDS》1989,3(2):133-138
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
11.
Mutational analysis supports a structural model for the cell cycle protein kinase p34 总被引:4,自引:0,他引:4
Endicott J. A.; Nurse P.; Johnson L. N. 《Protein engineering, design & selection : PEDS》1994,7(2):243-257
Structural models for the eukaryotic cell cycle control proteinp34 from human, S.pombe and S.cerevisiae have been derived fromthe crystallographic coordinates of the cAMP-dependent proteinkinase (cAPK) catalytic subunit (active conformation) and comparedwith the structure of Inactive CDK2 apoenzyme. Differences betweenthe p34 and cAPK catalytic sites provide a possible explanationfor their different substrate specificities. The p34 modelslocalize Tyrl5 and Thrl4 close to the sites of catalysis andsubstrate recognition where their phosphorylatlon could inhibitp34 kinase activity either by blocking MgATP or substrate binding.The conserved sequences PSTAIRE and LYLIFEFL are both closeto the catalytic site and accessible on the protein surfaceavailable to mediate interactions with other proteins. It ispredicted that p34 has an active-site cleft composed almostentirely of sequences common to all protein kinases and sequencesunique to the p34 protein family. Genetic and biochemical analysesof p34 have shown that it interacts extensively with a numberof other proteins. The model allows the relative dispositionof these sites of mutation to each other and to the sites ofcatalysis and substrate recognition to be appreciated. Surfaceregions on p34 that are important for function have been identified.These sites identify residues that may interact with p13SUCL,cydin, plO7WEEL and p80cdc25 相似文献
12.
In a systematic study of the periplasmic folding of antibodyfragments in Escherichia coli, we have analysed the expressionof an aggregation-prone and previously non-functional anti-phosphorylcholineantibody, T15, as a model system and converted it to a functionalmolecule. Introduction of heavy chain framework mutations previouslyfound to improve the folding of a related antibody led to improvedfolding of T15 fragments and improved physiology of the hostE.coli cells. Manipulation of the complementarity determiningregions (CDR) of the framework-mutated forms of T15 furtherimproved folding and bacterial host physiology, but no improvementwas seen in the wild type, suggesting the existence of a hierarchyin sequence positions leading to aggregation. Rational mutagenesisof the T15 light chain led to the production of functional T15fragments for the first time, with increased levels of functionalprotein produced from VH manipulated constructs. We proposethat a hierarchical analysis of the primary amino acid sequence,as we have described, provides guidelines on how correctly folding,functional antibodies might be achieved and will allow furtherdelineation of the decisive structural factors and pathwaysfavouring protein aggregation. 相似文献
13.
Finkeldei U.; Kalbitzer H. R.; Eisermann R.; Stewart G. C.; Hengstenberg W. 《Protein engineering, design & selection : PEDS》1991,4(4):469-473
The lactose-specific pbosphocarrier protein enzyme III of thebacterial phosphoenol-pyruvate-dependent phosphotransferasesystem of Staphylococcus aureus was modified by sitespecificmutagenesis on the corresponding lacF gene in order to replacethe histidine residues 78 and 82 of the amino acid sequencewith a serine residue. Wild-type and both mutant genes wereoverexpressed in Escherichia coli and the gene products werepurified to homogeneity. The conformation of wild-type and mutantproteins were monitored by 1H-NMR spectroscopy. In vitro phosphorylationstudies on mutant lactose-specific enzyme III, as well as evidencefrom NMR spectroscopy, lead to the conclusion that His78 isthe activesite for phosphorylation of lactose-specific enzymeIII by phospho-HPr (histidine-containing protein). The roleof His82 probably is the enhancement of velocity and efficiencyof the phosphotransfer from lactose-specific enzyme in to lactosespecifkenzyme II. This result refutes the conclusion of former workbased on data by protelytk cleavage and sequencing of the 32P-labeledpeptide of lactose-specific enzyme DTI that His82 is the active-sitefor phosphorylation. 相似文献
14.
Altering the association properties of insulin by amino acid replacement 总被引:12,自引:0,他引:12
Brems David N.; Alter Leila A.; Beckage Michael J.; Chance Ronald E.; DiMarchi Richard D.; Green L. Kenney; Long Harlan B.; Pekar Allen H.; Shields James E.; Frank Bruce H. 《Protein engineering, design & selection : PEDS》1992,5(6):527-533
The importance of ProB28 and LysB29 on the self-associationof insulin was established by systematically truncating theC terminus of the B chain. The relationship between structureand association was further explored by making numerous aminoacid replacements at B28 and B29 Association was studied bycircular dichroism, size-exclusion chromatography and ultracentrifugation.Our results show that the location of a prolyl residue at B28is critical for high-affinity self-association. Removal of ProB28in a series of C-terminal truncated insulins, or amino acidreplacement of Pro28, greatly reduced association. The largestdisruption to association was achieved by replacing LysB29 withPro and varying the amino acid at B28 Several of the analogswere predominantly monomers in solutions up to 3 mg/ml. Theseamino acid substitutions decreased association by primarilydisrupting the formation of dimers. Such amino acid substitutionsalso substantially reduced the Zn-induced insulin hexamer formation.The formation of monomeric insulins through amino acid replacementswas accompanied by conformational changes that may be the causefor decreased association. It is demonstrated that self-associationof insulin can be drastically altered by substitution of oneor two key amino acids. 相似文献
15.
Miura S Ferri S Tsugawa W Kim S Sode K 《Protein engineering, design & selection : PEDS》2008,21(4):233-239
Docking models of fructosyl amine oxidase (FAOD) from the marineyeast Pichia N1-1 (N1-1 FAOD) with the substrates fructosylvaline (f-Val) and fructosyl-N-lysine (f-Lys) were producedusing three-dimensional protein model as reported previously(Miura et al., 2006, Biotechnol. Lett., 28, 1895-1900). Theresidues involved in recognition of substrates were proposed,particularly Asn354, which interacts closely with f-Lys, butnot with f-Val. Substitution of Asn354 to histidine and lysinesimultaneously resulted in an increase in activity of f-valand a decrease in activity of f-Lys and thus, increasing thespecificity for f-Val from 13- to 19-fold. In addition to creatingtwo mutant FAODs with great potential for the measurement ofglycated hemoglobin, we have provided the first structural modelof substrate binding with eukaryotic FAOD, which is expectedto contribute to further investigation of FAOD. 相似文献
16.
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
17.
Finbow Malcolm E.; Eliopoulos Elias E.; Jackson Philip J.; Keen Jeffrey N.; Meagher Liam; Thompson Paul; Jones Philip; Findlay John B.C. 《Protein engineering, design & selection : PEDS》1992,5(1):7-15
A 16 kDa protein has been isolated in a homogeneous form asthe major component of a paracrystalline paired membrane structureclosely resembling the gap junction. The primary structure ofthis protein from arthropod and vertebrate species has beendetermined by protein and cDNA sequencing. The amino acid sequencesare highly conserved and virtually identical to the amino acidsequence of the proteolipid subunit of the vacuolar H+-ATPases.The disposition of the protein in the membrane has been studiedusing proteases and the N,N'-dicyclohexylcarbodiimide reactivesite identified. These data, together with secondary structurepredictions, suggest that the 16 kDa protein is for the mostpart buried in the membrane, arranged in a bundle of four hydrophobicß-helices. Using computer graphics, a model has beenconstructed based on this arrangement and on the electron microscopicimages of the paracrystalline arrays 相似文献
18.
Sloane David L.; Leung Regina; Barnett Jim; Craik Charles S.; Sigal Elliott 《Protein engineering, design & selection : PEDS》1995,8(3):275-282
Positional specificity determinants of human 15-lipoxygenasewere examined by site-directed mutagenesis and by kinetic analysisof the wild-type and variant enzymes. By comparing conserveddifferences among sequences of 12- and 15-lipoxygenases, a smallregion responsible for functional differences between 12- and15-lipoxygenases has been identified. Furthermore, the replacementof only two amino acids in 15-lipoxygenase (at 417 and 418 inthe primary sequence) by those found in certain 12-lipoxygenasesresults in an enzyme that has activity similar to 12-lipoxygenase.An examination of the activity of nine variants of lipoxygenasedemonstrated that the amino acid side-chain bulk and geometryof residues 417 and 418 are the key components of the positionalspecificity determinant of 15-lipoxygenase. Overexpression ofa variant (containing valines at positions 417 and 418) thatperforms predominantly 12-lipoxygenation was achieved in a baculovirus-insectcell culture system. This variant was purified to >90% homogeneityand its kinetics were compared with the wild-type 15-lipoxygenase.The variant enzyme has no change in its apparent KM for arachidonicacid and a minor(3-fold) change in its Vmax. For linoleic acid,the variant has no change in its KM and a 10-fold reductionin its Vmax, as expected for an enzyme performing predominantly12-lipoxygenation. The results are consistent with a model inwhich two amino acids of 15-lipoxygenase (isoleucine 417 andmethionine 418) constitute a structural element which contributesto the regiospecificity of the enzyme. Replacement of theseamino acids with those found in certain 12-lipoxygenases resultsin an enzyme which can bind arachidonic acid in a catalyticregister that prefers 12-lipoxygenation. 相似文献
19.
Interactions of (Ala*Ala*Lys*Pro)n and (Lys*Lys*Ser*Pro)n with DNA. Proposed coiled-coil structure of AlgR3 and AlgP from Pseudomonas aeruginosa 总被引:1,自引:0,他引:1
Medvedkin Vyacheslav N.; Permyakov Eugene A.; Klimenko Lyubov V.; Mitin Yuri V.; Matsushima Norio; Nakayama Susumu; Kretsinger Robert H. 《Protein engineering, design & selection : PEDS》1995,8(1):63-70
The proteins, AlgR3 and AlgP, are involved in the regulationof alginate synthesis in Pseudomonas. They contain multiplerepeats of Ala*Ala*Lys*Pro as do several other proteins thatresemble histones. The interactions of synthesis oligopeptidescomposed of repeated Ala*Ala*Lys*Pro or Lys*Lys*Ser*Pro unitswith DNA were studied by fluorescence of the Fmoc (9-fluorenylmethyloxycarbonyl)group attached to the N-termini of the peptides. DNA quenchingof the Fmoc fluorescence of the peptides was used to estimatethe apparent association constants for the interaction of Fmoc(AAKP)nOH(n = 2, 4, 8, 18, 32) and of Fmoc(KKSP)nOH (n = 2, 4, 8, 16,20, 32) with DNA. The Fmoc(AAKP)nOH peptides bind to DNA onlyat low ionic strength; the Fmoc(KKSP)n OH peptides interactwith DNA at both low (0.05 M KCl) and high (0.2 M KCl) saltAt low ionic strength an increase in the number of the repeatunits causes an increase in the apparent association constantup to {small tilde}2 x 106 M1 for both types of peptidesat N 24. The insertion of an AAKTA unit into the middle ofthe Fmoc(AAKP)8OH peptide increases its affinity to DNA. Wepropose a model of (AAKP)n and of its interaction with DNA.The repeat unit consists of a single turn of -helix followedby a bend necessitated by Pro. The resultant coiled-coil formsa right-handed superhelix with 10 AAKPs per repeat distanceof {small tilde}33 Å. With only slight modification ofthe canonical parameters of this model the AAKP super helixfits into the major groove of B-form DNA with one AAKP tetramerper base pair repeat of 3.4 Å. The -amine nitrogen ofLys can form a polar hydrogen bond with a phosphate oxygen atomof the DNA backbone. A better fit is obtained when the modelis modified to accommodate [(AAKP)5AAKTA]n as actually observedin AlgR3. We suggest that this coiled-coil represents a generalmotif for other proteinDNA interactions. 相似文献
20.
A pore-forming protein with a metal-actuated switch 总被引:4,自引:0,他引:4
Walker Barbara; Kasianowicz John; Krishnasastry Musti; Bayley Hagan 《Protein engineering, design & selection : PEDS》1994,7(5):655-662
Staphylococcal -hemolysin, a pore-forming exotoxin, is a polypeptideof 293 amino acids that is secreted by Staphylococcus aureusas a water-soluble monomer. It assembles to form hexameric poresin lipid bilayers. Previous studies of pore formation have establishedthe involvement of a central glycine-rich loop. Here, we showthat when five consecutive histidine residues replace aminoacids 130134 at the midpoint of the loop, they providea switch with which pore activity can be (i) turned off by micromolarconcentrations of divalent zinc ions and (ii) turned back onwith the chelating agent EDTA. Planar bilayer recordings showthat Zn2+ and EDTA can act on open channels from either sideof the bilayer and thus demonstrate that the central loop linespart of the conductive pathway. Our results suggest that genetically-engineeredpore-forming proteins might make useful components of metalion sensors 相似文献