共查询到20条相似文献,搜索用时 46 毫秒
1.
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
2.
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
3.
Luger Karolin; Szadkowski Halina; Kirschner Kasper 《Protein engineering, design & selection : PEDS》1990,3(4):249-258
Protein sequences containing redundant segments of secondarystructure at both termini have the choice a priori of foldinginto several possible circularly permuted variants of the wild-typetertiary structure. To test this hypothesis the gene of phosphoribosylanthranilate isomerase from yeast, which is a single-domain8-fold ß barrel protein, was modified to produce a10-fold ß homologue in Escherichia coli. It containeda duplicate of the two C-terminal ß units of supersecondarystructure fused to its N-terminus. Most of the protein was recoveredfrom the insoluble fraction of disrupted cells by dissolutionin guanidinium chloride solutions and refolding. Pristine proteinwas purified from the soluble fraction. The purified (ß)10proteins were enzymically almost fully active. Absorbance, fluorescenceand circular dichroism spectra as well as the reversible unfoldingbehaviour of both proteins were also very similar to the propertiesof the original (ß)8 protein. Digestion with endopeptidasesconverted both the pristine and the refolded (ß)10variant to the same large fragment that had the N-terminal sequenceand mol. wt of the wild-type ß)8 protein. The datasuggest that the folding of the (ß)10 variant is controlledthermodynamically both in vivo and in vitro. 相似文献
4.
Using a series of homologous calcium-binding proteins, a quantitativestructureactivity relationship (QSAR), log(1/Kd) = 18.986 1.6278(X1) + 0.7981(X2) + 0.2312(X3), has been established,which relates the calcium-binding affinities (1/Kd) of the regulatoryproteins with (i) the net ligand charge (X1) of the two calciumbinding loops, (ii) the hydrophobicity (X2) of the ß-sheetsegment of the loops and (iii) the hydrophobicity (X3) of thefour EF-hand helices. It is found that the bindingaffinities are influenced by the EF-hand pairrather than the individual EF-hands. The QSAR,in addition to explaining satisfactorily the large variationin the observed calcium affinities, can predict the affinitiesof the EF-hand pairs in other proteins from theamino acid sequence and can also account for the changes inthe affinities caused by substitution in the hydrophobic and/ormetal-coordinating residues. Thus, this relationship can beemployed in protein design and engineering. The method is potentiallyuseful in the development of similar relationships for the bindingof other proteins to substrates, inhibitors, drugs and co-factors. 相似文献
5.
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
6.
Witt Ellen; Stewart George C.; Hengstenberg Wolfgang 《Protein engineering, design & selection : PEDS》1992,5(3):267-271
The lacG gene encoding the 6-phospho-ß-galactosidase(E.C.3.2.1.85) of Staphylococcus aureus was fused to the proteinA gene in the plasmid pRIT2T. Escherichia coli cells containingthis plasmid produce a fusion protein with both IgG bindingand 6-phospho-ß-galactosidase activities after heatinduction. The recombinant gene was overexpressed and the hybridprotein was purified to homogeneity in high yield. The chimericprotein was shown to have almost identical enzymatic characteristicsto pure 6-phospho-ß-galactosidase. This result leadsto the conclusion that a free N-terminus of the 6-phospho-ß-galactosidaseis not required for biological activity. The hybrid proteinof protein A and 6-phospho-ß-galactosidase was usedas an enzyme conjugate in enzyme-linked immunosorbent assays(ELISA). The experiments presented demonstrate that the 6-phospho-ß-galactosidaseis a suitable fusion partner in various diagnostic applicationswhere an unique biological activity is required. 相似文献
7.
Rypniewski W.R.; Hastrup S.; Betzel Ch.; Dauter M.; Dauter Z.; Papendorf G.; Branner S.; Wilson K.S. 《Protein engineering, design & selection : PEDS》1993,6(4):341-348
The trypsin from Fusarium oxysporum is equally homologous totrypsins from Streptomyces griseus, Streptomyces erythraeusand to bovine trypsin. A DFP (diisopropylfluorophosphate) inhibitedform of the enzyme has been crystallized from 1.4 M Na2SO4,buffered with citrate at pH 5.05.5. The crystals belongto space group P21 with cell parameters a=33.43 Å, b=67.65Å, c=39.85 Å and ß=107.6°. There isone protein molecule in the asymmetric unit. X-ray diffractiondata to a resolution of 1.8 Å were collected on film usingsynchrotron radiation. The structure was solved by molecularreplacement using models of bovine and S.griseus trypsins andrefined to an R-factor of 0.141. The overall fold is similarto other trypsins, with some insertions and deletions. Thereis no evidence of the divalent cation binding sites seen inother trypsins. The covalently bound inhibitor molecule is clearlyvisible. 相似文献
8.
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
9.
Copeland William C.; Vanderslice Peter; Robertus Jon D. 《Protein engineering, design & selection : PEDS》1987,1(5):419-423
The genes coding for histidine decarboxylase from a wild-typestrain and an autoactivation mutant strain of Lactobacillus30a have been cloned and expressed in Escherichia coli. Themutant protein, G58D, has a single Asp for Gly substitutionat position 58. The cloned genes were placed under control ofthe ß-galactosidase promoter and the products arenatural length, not fusion proteins. The enzyme kinetics ofthe proteins isolated from E. coli are comparable to those isolatedfrom Lactobacillus 30a. At pH 4.8 the Km of wild-type enzymeis 0.4 mM and the kcat = 2800 min1; the correspondingvalues for G58D are 0.5 mM and 2750 min1. The wild-typeand G58D have autoactivation half-times of 21 and 9 h respectivelyunder pseudophysiological conditions of 150 mM K+ and pH 7.0.At pH 7.6 and 0.8 M K+ the half times are 4.9 and 2.9 h. Therelatively slow rate of autoactivation for purified proteinand the differences in cellular and non-cellular activationrates, coupled with the fact that wild-type protein is readilyactivated in wild-type Lactobacillus 30a but poorly activatedin E. coli, suggest that wild-type Lactobacillus 30a containsa factor, possibly an enzyme, that enhances the activation rate. 相似文献
10.
Wu Zhen; Demma Mark; Strickland Corey L.; Syto Rosalinda; Le Hung V.; Windsor William T.; Weber Patricia C. 《Protein engineering, design & selection : PEDS》1999,12(4):341-348
Protein farnesyltransferase (FPT) is a 97 000 Da heterodimericenzyme that catalyzes post-translational farnesylation of manycellular regulatory proteins including p21 Ras. To facilitatethe construction of site-directed mutants, a novel translationallycoupled, two-cistron Escherichia coli expression system forrat FPT has been developed. This expression system enabled yieldsof >5 mg of purified protein per liter of E.coli cultureto be obtained. The E.coli-derived FPT demonstrated an activitycomparable to that of protein isolated from other sources. Thereported expression system was used to construct three ß-subunitC-terminal truncation mutants, 5, 10 and 14, which were designedto eliminate a lattice interaction between the ß-subunitC-terminus of one molecule and the active site of a symmetry-relatedmolecule. Steady-state kinetic analyses of these mutants showedthat deletion up to 14 residues at the C-terminus did not reducethe value of kcat; however, Km values for both peptide and FPPincreased 23-fold. A new crystalline form of FPT was obtainedfor the 10 C-terminal mutant grown in the presence of the substrateanalogs acetyl-Cys-Val-Ile-Met-COOH peptide and -hydroxyfarnesylphosphonicacid. The crystals diffract to beyond 2.0 Å resolution.The refined structure clearly shows that both substrate analogsadopt extended conformations within the FPT active site cavity. 相似文献
11.
Kim Hyo Joon; Nishikawa Satoshi; Tanaka Toshiki; Uesugi Seiichi; Takenaka Hitoshi; Hamada Minoru; Kuby Stephen A. 《Protein engineering, design & selection : PEDS》1989,2(5):379-386
An artificial gene coding for the human muscle-type cytosolicadenylate kinase (hAK1) was chemically synthesized and directlyexpressed in Escherichia coli under the control of trp promoter.The DNA duplex of 596 bp was designed and constructed from 40oligonucleotide fragments of typically 30 nucleotides in length.Twelve unique restriction sites were fairly evenly spaced inthe synthetic gene to facilitate site-specific mutagenesis atany part of this recombinant protein. The genes for mutant hAK1(Tyr 95 Phe 95, Y95F hAK1; Arg 97 Ala 97, R97AhAK1) were constructed by cassette mutagenesis and utilizedrestriction sites incorporated in the hAK1 gene. The recombinanthAK1 was purified to homogeneity by a two-step chromatographicprocedure with a good yield, and showed the same adenylate kinaseactivity as that of authentic hAK1. preliminary kinetic studiesshow that the enzymatic activity (Vmax app,cor) 相似文献
12.
Greenwood M.Jeffrey; Ong Edgar; Gilkes R.Neil; Warren R.Antony J.; Miller C.Robert Jr; Kilburn G.Douglas 《Protein engineering, design & selection : PEDS》1992,5(4):361-365
The endoglucanase CenA and the exoglucanase Cex from Cellulomonasfimi each contain a discrete cellulose-binding domain (CBD),at the amino-terminus or carboxyl-terminus respectively. Thegene fragment encoding the CBD can be fused to the gene of aprotein of interest. Using this approach hybrid proteins canbe engineered which bind reversibly to cellulose and exhibitthe biological activity of the protein partner. Alkaline phosphatase(PhoA) from Escherichia coli, and a ß-glucosidase(Abg) from an Agrobacterium sp. are dimeric proteins. The fusionpolypeptides CenA-PhoA and Abg-CBCcex are sensitive to proteolysisat the junctions between the fusion partners. Proteolysis resultsin a mixture of homo- and heterodimers; these bind to celluloseif one or both of the monomers carry a CBD, e.g. CenA-PhoA/CenA-PhoAand CenA-PhoA/PhoA. CBD fusion polypeptides could be used inthis way to purify polypeptides which associate with the fusionpartner. 相似文献
13.
Witt Ellen; Frank Rainer; Hengstenberg Wolfgang 《Protein engineering, design & selection : PEDS》1993,6(8):913-920
The 6-phospho-ß-galactosidase of Staphylococcus aureus,Lactococcus lactis and Lactobacillus casei and 6-phospho-ßglucosidaseB of Escherichia coli build a subfamily inside a greater enzymefamily, named the glycosal hydrolase family 1, which, hi addition,contains nine ß-glycosidases of different origins.Kinetic and immunological evidence is provided in this reportwhich strengthens the relationship of the four 6-phospho-ß-glycosidases.It is shown that the 6-phospho-ß-galactosidases and6-phospho-ß-glucosidase B are able to split aromaticß-galactoside phosphates and ß-glucosidephosphates. The turnover numbers of hydrolysis of substrateswith different epimerization at C-4 of the glycon vary up to15-fold only. Two polydonal antisera, one derived against thenative 6-phospho-ß-galactosidase from S.aureus andthe other derived against the 6-phospho-ß-glucosidaseB, cross-reacted with both enzymes. Peptides of the proteinswere separated by reverse phase HPLC. The cross-reacting peptideswere sequenced and shown to be localized at almost the sameposition in the aligned primary structures of both enzymes.An insertion of nine amino adds near these antigenic domainsis unique for the 6-phospho-ß-glycosidases and missingwithin the sequences of the ß-glycoside-specific membersof the family. The lacG gene of a 6-phospho-ß-galactosidasenegative S.aureus mutant was doned into E.coli and sequenced.In the totally inactive mutant protein only the glycine at position332 was changed to an arginine. This amino acid is part of thesequence insertion near the antigenic domain reacting with bothantisera. These data support the assumption that the regionis of great importance for the function of the enzymes and thatit is possible it determines the specificity of the phosphorylatedform of the substrates. In addition, the 6-phospho-ß-galactosidaseof S.aureus was modified by sitedirected mutagenesis of thecorresponding lacG gene hi order to replace residues Glul60and Glu375, which were suspected of being involved hi the generalacid catalysis of substrate hydrolysis, with glutamine residues.The mutant protein 160EQ retained some catalytic activity whilethe protein 375EQ was totally inactive. Glu375 is the activesite nudeophile of the 6-phospho-ß-galactosidase ofS.aureus. It is located in the sequence motif ENG where Glu358was identified as the catalytkally active nudeophile hi theß-glucosidase of Agrobacterium. 相似文献
14.
Smallshaw Joan E.; Georges Fawzy; Lee Jeremy S.; Waygood E.Bruce 《Protein engineering, design & selection : PEDS》1999,12(7):623-630
The monoclonal antibody Jel42 is specific for the Escherichiacoli histidine-containing protein, HPr, which is an 85 aminoacid phosphocarrier protein of the phosphoenolpyruvate:sugarphosphotransferase system. The binding domain (Fv) has beenproduced as a single chain Fv (scFv). The scFv gene was synthesizedin vitro and coded for pelB leader peptideheavy chainlinkerlightchain(His)5 tail. The linker is three repeats from theC-terminal repetitive sequence of eukaryotic RNA polymeraseII. This linker acts as a tag; it is the antigen for the monoclonalantibody Jel352. The codon usage was maximized for E.coli expression,and many unique restriction endonuclease sites were incorporated.The scFv gene incorporated into pT7-7 was highly expressed,yielding 1030% of the cell protein as the scFv, whichwas found in inclusion bodies with the leader peptide cleaved.Jel42 scFv was purified by denaturation/renaturation yieldingpreparations with Kd values from 20 to 175 nM. However, basedupon an assessment of the amount of active refolded scFv, thebinding dissociation constant was estimated to be 2.7 ±2.0 nM compared with 2.8 ± 1.6 and 3.7 ± 0.3 nMpreviously determined for the Jel42 antibody and Fab fragmentrespectively. The effect of mutation of the antigen HPr on thebinding constant of the scFv was very similar to the propertiesdetermined for the antibody and the Fab fragment. It was concludedthat the small percentage (~6%) of refolded scFv is a true mimicof the Jel42 binding domain and that the incorrectly foldedscFv cannot be detected in the binding assay. 相似文献
15.
Smith D.D.S.; Pratt K.A.; Sumner I.G.; Henneke C.M. 《Protein engineering, design & selection : PEDS》1995,8(1):13-20
A protein designed de novo to fold into the Greek key jellyrollstructural motif has been studied. Theoretical analyses haveindicated that the designed sequence should adopt the ß-strandarrangement of the Greek key jellyroll rather than any otherarrangement. A synthetic gene was constructed and the proteinexpressed in Escherichia coli. Circular dichroism spectroscopyis consistent with the protein folding into the designed conformationand also suggests the presence of tertiary structure. Fluorescencespectroscopy showed the single tryptophan to be partially buried,while denaturation studies showed changes in fluorescence toprecede alterations in secondary structure. 相似文献
16.
Blanchin-Roland Sylvie; Masson Jean-Michel 《Protein engineering, design & selection : PEDS》1989,2(6):473-480
The inability of Escherichia coli to secrete proteins in growthmedium is one of the major drawbacks in its use in genetic engineering.A synthetic gene, homologous to the one coding for the kil peptideof pColE1, was made and cloned under the control of the lacpromoter, in order to obtain the inducible secretion of homologousor heterologous proteins by E.coli. The efficiency of this syntheticgene to promote secretion was assayed by analysing the productionand secretion of two proteins, the R-TEM1 ß-lactamase,and the -amylase from Bacillus licheniformis. This latter proteinwas expressed in E.coli from its gene either on the same plasmidas the kil gene or on a different plasmid. The primary effectof the induction of the kil gene is the overproduction of thesecreted proteins. When expressed at a high level, the kil genepromotes the overproduction of all periplasmic proteins andthe total secretion in the culture medium of both the ß-lactamaseor the -amylase. This secretion is semi-selective for most periplasmkproteins are not secreted. The kil peptide induces the secretionof homologous or heterologous proteins in two steps, first actingon the cytoplasmic membrane, then permeabilizing the outer membrane.This system, which is now being assayed at the fermentor scale,is the first example of using a synthetic gene to engineer anew property into a bacterial strain. 相似文献
17.
Dabrowski Michael J.; Dietze Eric C.; Atkins William M. 《Protein engineering, design & selection : PEDS》1996,9(3):291-298
Escherichia coli glutamine synthetase (GS) is a dodecamer ofidentical subunits which are arranged as two face-to-face hexamericrings. In the presence of 10% ammonium sulfate, wild type GSexhibits a pH-dependent salting out with a pKaof 4.51. Electron micrographs indicate that the pH-dependentaggregation corresponds to a highly specific self-assembly ofGS tubules, which result from stacking of individual dodecamers.This stacking of dodecamers is similar to the metal ion-inducedGS tubule formation previously described. Site-directed mutagenesisexperimentsindicate that the N-terminal helix of each subunit is involvedin the salting out reaction, as it is in the metal-induced stacking.A single substitution of alanine for His4 completely abolishesthe (NH42SO4-induced aggregation. However, the H4C mutant proteindoes nearly completely precipitate under the same salting outconditions. Mutations at other residues within the helix haveno effect on the stacking reaction. Differential catalyticactivityof unadenylylated GS versus adenylylated GS has been used todetermine whether wild type dodecamers complementthe H4A mutant in the stacking reaction. The complementationexperiments indicate that His4 residues on bothsides of thedodecamer-dodecamer interfaces are not absolutely required forsalting out, although the wild type dodecamers clearly stackpreferentially with other wild type dodecamers. Approximately20% of the protein precipitated fromthe mixtures containingthe wild type GS and the H4A mutant is the mutant. The implicationsof these results for protein engineering are discussed. 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.
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
20.
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