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1.
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
2.
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
3.
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
4.
Siezen Roland J.; Bruinenberg Paul G.; Vos Pieter; Alen-Boerrigter Ingrid Van; Nijhuis Monique; Alting Arno C.; Exterkate Fred A.; Vos Willem M.de 《Protein engineering, design & selection : PEDS》1993,6(8):927-937
The substrate-binding region of the cell-envelope proteinaseof Lactococcus lactis strain SK11 was modelled, based on sequencebomology of the catalytic domain with the serine proteinasessubtilisin and thermitase. Substitutions, deletions and insertionswere introduced, by site-directed and cassette mutagenesfe ofthe prtP gene encoding this enzyme, based on sequence comparisonboth with subtilisin and with the homologous L.lactis strainWg2 proteinase, which has different proteolytic properties.The engineered enzymes were investigated for thermal stability,proteolytic activity and cleavage specificity towards smallchromogenk peptide substrates and the peptide g1-casein(l23).Mutations in the subtilisin-like substrate-binding region showedthat Ser433 is the active site residue, and that residues 138and 166 at either side of the binding cleft play an importantrole in substrate specificity, particularly when these residuesand the substrate are oppositely charged. The K748T mutationin a different domain also affected specificity and stability,suggesting that this residue is in close proximity to the subtilisin-likedomain and may form part of the substratebinding site. Severalmutant SK11 proteinases have novel properties not previouslyencountered in natural variants. Replacements of residues 137139AKTalong one side of the binding cleft produced the 137139GPPmutant proteinase with reduced activity and narrowed specificity,and the 137139GLA mutant with increased activity andbroader specificity. Furthermore, the 137139GDT mutanthad a specificity towards g1,-casein(l23) closely resemblingthat of L.lactis Wg2 proteinase. Mutants with an additionalnegative charge in the binding region were more stable towardsautoproteolysis. 相似文献
5.
Heaphy Shaun; Singh Mohinder; Gait Michael J. 《Protein engineering, design & selection : PEDS》1987,1(5):425-431
A synthetic gene coding for the bacteriocidal protein caltrin/seminalplasminwas constructed and expressed in Escherichia coli as a fusionwith ß-galactosidase. The gene was designed with arecognition site for the plasma protease, Factor Xa, coded forimmediately prior to the N-terminus of caltrin. The ß-galactosidase-caltrinfusion protein was cleaved with Factor Xa to give caltrin, whichwas identified by its size on SDS-PAGE, its ability to reactwith an antiserum raised to the N-terminal nonapeptide of caltrinand its N-terminal amino acid sequence. After partial purification,synthetic caltrin was found to be active in an assay involvinginhibition of growth of E.coli. 相似文献
6.
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
7.
The facile detection and purification of a recombinant proteinwithout detailed knowledge about its individual biochemicalproperties constitutes a problem of general interest in proteinengineering. The use of a novel kind of random peptide libraryfor the stepwise engineering of a C-terminal fusion peptidewhich confers binding activity towards streptavidin is describedin this study. Because of its widespread use as part of a varietyof conjugates and other affinity reagents, streptavidin constitutesthe binding partner of choice both for detection and purificationpurposes. The streptavidin-affinity tag was engineered at theC-terminus of the VH domain as part of the D1.3 Fv fragmentwhich was functionally expressed in Escherichia coli. Irrespectiveof whether it was displayed by the VH or the VL domain, theoptimized version of the affinity peptide termed Strep-tagallowed the detection of the Fv fragment both on Western blotsand in ELISAs by a streptavidinalkaline phosphatase conjugate.In addition, the one-step purification of the intact Fv fragmentcarrying a single Strep-tag at the C-terminus of only one ofits domains was achieved by affinity chromatography with streptavidin-agaroseusing very mild elution conditions. 相似文献
8.
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
9.
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
10.
Kurasaki Masaaki; Emoto Tadasu; Arias Ana Rosa Linde; Okabe Masashi; Yamasaki Futoshi; Oikawa Shinji; Kojima Yutaka 《Protein engineering, design & selection : PEDS》1996,9(12):1173-1180
We examined the independent self-assembly of the - and ß-fragmentsof human metallothionein (MT) into cadmiumbinding conformationin an Escherichia coli expression system, in addition to wild-typeMT expression. The expressed -fragment formed independentlythe structure of a metal-binding cluster without the aid ofthe ß-fragment. The -fragment and wild-type MT expressedin E.coli were purified and analyzed for their biochemical andspectroscopic properties. The apparent cadmium binding of the-fragment was approximately 12-fold greater than that for thewild-type MT, whereas in other respects the studied biochemicalproperties were similar. In contrast, we were unable to obtainany independently expressed ß-fragment as the cadmium-bindingform in this study. Possible explanations for this phenomenonare discussed. 相似文献
11.
Goth Cynthia R.; Loh Chong-Seng; Porter Alan G. 《Protein engineering, design & selection : PEDS》1991,4(7):785-791
Single amino acid substitutions were generated in predictedhydrophilic loop regions of the human tumour necrosis factorbeta (TNF-ß) molecule, and the mutant proteins wereexpressed in Escherichia coli and purified. Mutants with singleamino acid changes at either of two distinct loop regions, atpositions aspartic acid 50 or tyrosine 108, were found to havegreatly reduced receptor binding and cytotoxic activity. Thesetwo regions in TNF-ß correspond to known loop regionswhere mutations also result in loss of biological activity ofTNF, a related cytokine which shares the same cellularreceptors with TNF-ß. The two distinct loops at positions31-34 and 84-89 in the known three-dimensional structure ofTNF- (equivalent to positions 4650 and 105110respectively in TNF-ß), lie on opposite sides of theTNF- monomer. When the TNF-a monomer forms a trimer, the twoloops, each from a different subunit of the trimer, come togetherand lie in a cleft between adjacent subunits. Together, thesefindings suggest that a TNF receptor binds to a cleft betweensubunits via surface loops at amino acid residues 3134and 8489 in TNF, and similarly via surface loopsincluding amino acids aspartic acid 50 and tyrosine 108 in TNFß. 相似文献
12.
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
13.
Adib-Conquy Minou; Gilbert Michele; Christodoulou Christina; Avrameas Stratis 《Protein engineering, design & selection : PEDS》1995,8(9):859-863
In this report, we describe the expression system that enabledus to produce in Escherichia coli the Fab fragment of a mouseIgM that has previously been shown to inhibit the binding ofIgG to autoantigens by interacting with their variable regions.In our system, both light chain and heavy chain fragments wereput under the control of the malE promoter. The light chainwas fused to the MalE signal sequence, while the heavy chainvariable and first constant region were fused to the alkalinephosphatase signal sequence. In this system, after inductionof the promoter with maltose, the Fab fragment could be detectedin a periplasmic extract of the bacteria by Western blottingand also by ELISA. This Fab fragment was purified on a goatanti-mouse immunoglobulin immunoadsorbent and biotinylated.The Fab fragment produced by E.coli reacted with the trinitrophenyl(TNP) hapten and F(ab')2 fragments of mouse IgG and these reactivitiescould be specifically inhibited by the corresponding solubleantigens. The dissociation constants of this Fab were 1.65 x106 M for TNP and 5 x 106 M for IgG F(ab')2 fragments,indicating that the affinity of the Fab fragment compared withthat of the whole IgM molecule was similar for TNP but was lowerfor IgG F(ab')2 fragments 相似文献
14.
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
15.
Przybycien Todd M.; Dunn Joseph P.; Valax Pascal; Georglou Georage 《Protein engineering, design & selection : PEDS》1994,7(1):131-136
The secondary structure of proteins in E.coli inclusion bodieswas investigated via Raman spectroscopy. Inclusion bodies werepurified from cells expressing different forms of RTEM ß-lactamaseand grown at either 37 or 42° C. All of the solid phaseinclusion body samples examined gave amide I band spectra thatwere perturbed from that of the native, purified protein inboth solution and powder forms; secondary structure estimatesindicated significant decreases in a-helix and increases inß-sheet contents in the inclusion body samples. The structureestimates for inclusion bodies isolated from 37°C cultureswere similar, regardless of aggregate localization in the E.colicytoplasmic or periplasmic spaces or ß-lactamase precursorcontent. Inclusion bodies obtained from 42°C cells exhibiteda further reduction of ß-helix and augmentation of ß-sheetcontents relative to those from 37°C cultures. These resultsare consistent with the paradigm for inclusion body formationvia the self-association of intra-cellular folding intermediateshaving extensive secondary structure content. Further, the overallsecondary structure content of inclusion bodies is not significantlyaffected by subcellular compartmentalization, but may be alteredat increased temperatures 相似文献
16.
Making tissue-type plasminogen activator more fibrin specific 总被引:2,自引:0,他引:2
Paoni Nicholas F.; Chow Alice M.; Pena Luis C.; Keyt Bruce A.; Zoller Mark J.; Bennett William F. 《Protein engineering, design & selection : PEDS》1993,6(5):529-534
The fibrin specificity of tissue-type plasminogen activatorcan be increased by mutagenesis within at least four sites inthe protease domain. These sites include residue I276, the newN-terminus formed by conversion to a two-chain structure, residueson either side of the active site cleft, KHRR 296299or DDD 364366, a charged surface involved in fibrin interactions,which includes residues H432, R434, D460, R462 and a loop structure,PQANL 466470, near the fibrin-binding patch. Variantswith mutations at any of these sites have low fibrinogen-stimulatedactivity, whereas fibrin-stimulated activity is at least normal.Kinetic analysis reveals that mutations at these positions reducethe kcat in the presence of fibrinogen, but leave the moleculeswith normal kinetic constants in the presence of fibrin. A significantexception is found at positions 296299, where the presenceof fibrin manifests significant increases in both kcat and Km.Combinations of mutations at these sites appear to be additivewith respect to fibrin specificity. 相似文献
17.
Peter W.; Dunkel R.; Stouten P.F.W.; Vriend G.; Beato M.; Suske G. 《Protein engineering, design & selection : PEDS》1992,5(4):351-359
The progesteronebinding protein uteroglobin has beenexpressed in Escherichia coli in an unfused, soluble form. likemature uteroglobin from rabbit endometrium (UG), the E.coliproduceduteroglobin (UG1) dimerizes in vitro, forms an antiparalleldimer with Cys3Cys69' and Cys69Cys3' disulfidebonds and binds progesterone under reducing conditions. In orderto analyze the dimerization and the reduction dependence ofprogesterone binding in more detail, we separately replacedcysteine 3 and cysteine 69 by serines. Under reducing conditions,both uteroglobin variants (UGl3Ser and UGl69Ser)bind progesterone with the same affinity as the wildtypesuggesting that both cysteine residues are not directly involvedin progesterone binding. In contrast to the wildtypeprotein, both cysteine variants also bind progesterone withhigh affinity in the absence of reducing agents. In addition,UGl-3Ser and UGl-69Ser both form covalently linked homodimers.Thus, unnatural Cys6969' and Cys33' disulfidebonds exist in UG13Ser and UG169Ser, respectively.These data together with computer models based on X-ray diffractiondata strongly support the idea that progesterone reaches itsbinding site located in an internal hydrophobic cavity via ahydrophobic tunnel along helices 1 and 4. Under nonreducingconditions the tunnel is closed by two disulfide bridges (Cys3Cys69'(and Cys69Cys3') that lie in the most flexible regionof the dimer. Reduction or replacement of a cysteine residueenables conformational changes that open the channel allowingprogesterone to enter. 相似文献
18.
Hefford Mary Alice; Laderoute Keith; Willick Gordon E.; Yaguchi Makoto; Seligy Verner L. 《Protein engineering, design & selection : PEDS》1992,5(5):433-439
The C-terminal boundary of primary sequence of the Bacillussubtilis PAP115 endo-ß-1,4-glucanase (EG) requiredfor stable catalytic activity has been mapped by site-directedmutagenesis using Escherichia coli as host. The 52 kDa cel geneproduct, EG470 and a 33 kDa mutant (EG300), lacking 170 residuesthrough a nonsense mutation at the leucine-330 codon of thegene, exhibited similar patterns of enzymatic activity and pHoptima using cellooligopentaose as substrate.CD spectra indicatedthat the bulk of the -helical secondary structure in EG470 wascontained within EG300. However, relative to EG470, the specificactivity of EG300 was 3- to 4-fold lower with amorphous celluloseas substrate and {small tilde}4-to5-fold higher with carboxymethylcellulose(soluble cellulose).These results along with data which showthat EG470 binding capacity to mirocrystalline cellulose is{small tilde} 11 times more than that of EG300, demonstratethe importance of residues 330499 for non-catalytic bindingof cellulose. A construct of the cel gene carrying a deletionof codons 330499 and an insertion of a nonsense codonat leucine-330, was further used to make mutants EG296 and EG291with nonsense codon substitutions at arginine and serine-321,respectively.Western analysis using EG-specific antiserum revealedthat relative losses in enzymatic activity of EG296 (50%) andEG291 (95%) could be accounted for by the extent of their proteolysis,signifying a marked destabilization of these enzymes by removalof only a few amino acids. 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.
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