共查询到20条相似文献,搜索用时 620 毫秒
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Nakazawa Takashi; Kawai Hikaru; Okamoto Yuko; Fukugita Masataka 《Protein engineering, design & selection : PEDS》1992,5(6):495-503
A tertiary structure prediction is described using Monte Carlosimulated annealing for the peptide fragment corresponding toresidues 1636 of bovine pancreatic trypsin inhibitor(BPTI). The simulation starts with randomly chosen initial conformationsand is performed without imposing experimental constraints usingenergy functions given for generic interatomic interactions.Out of 20 simulation trials, seven conformations show a sheet-likestructuretwo strands connected by a turnalthoughthis sheet-like structure is not as rigid as that observed innative BPTI. It is also shown that these conformations are mostlylooped and exhibit a native- like right-handed twist. Unlikethe case with the C-peptide of RNase A, no conspicuous -helicalstructure is found in any of the final conformations obtainedin the simulation. However, the lowest-energy conformation doesnot resemble exactly the native structure. This indicates thatthe rigid ß-sheet conformation of native BPTI merelycorresponds to a local minimum of the energy function if thefragment with residues 1636 is isolated from the nativeprotein. A statistical analysis of all 20 final conformationssuggests that the tendency for the peptide segments to formextended ß-strands is strong for those with residues1824, and moderate for those with residues 3035.The segment of residues 2529 does not tend to form anydefinite structure. In native BPTI, the former segments areinvolved in the ß-sheet and the latter in the turn.A folding scenario is also speculated from this analysis. 相似文献
3.
Hirst Jonathan D.; Vieth Michal; Skolnick Jeffrey; Brooks Charles L. III 《Protein engineering, design & selection : PEDS》1996,9(8):657-662
The leucine zipper structure is adopted by one family of thecoiled coil proteins. Leucine zippers have a characteristicleucine repeat: LeuX6LeuX6LeuX6Leu(where X may be any residue). However, many sequences have theleucine repeat, but do not adopt the leucine zipper structure(we shall refer to these as non-zippers). We have found andanalyzed residue pair patterns that allow one to identify correctly90% of leucine zippers and 97% of non-zippers. Simpler analyses,based on the frequency of occurrence of residues at certainpositions, specify, at most, 65% of zippers and 8090%of non-zippers. Both short and long patterns contribute to thesuccessful discrimination of leucine zippers from non-zippers.A number of these patterns involve hydrophobic residues thatwould be placed on the solvent-exposed surface of the helix,were the sequence to adopt a leucine zipper structure. Thus,an analysis of protein sequences has allowed us to improve discriminationbetween leucine zippers and non-zippers, and has provided somefurther insight into the physical factors influencing the leucinezipper structure. 相似文献
4.
Nakatani Tomoyuki; Lone Yu-Chun; Yamakawa Junko; Kanaoka Masaharu; Gomi Hideyuki; Wydenes John; Noguchi Hiroshi 《Protein engineering, design & selection : PEDS》1994,7(3):435-443
Mouse monoclonal antihuman IL2 receptor antibody(BB10) inhibits EL2dependent human Tcellproliferation. It has been used in clinical trials in the transplantationfield and promising results are being accumulated. Mouse BB10antibody was humanized by grafting all CDRs and some frameworkamino add residues onto human antibodies, KAS for VH and PAYfor Vx. Nine humanized BBlOs with differently graftedframework residues were constructed and assessed for their biologicalactivities. One of these humanized BB10, M5, showed nearlythe same activity as the mouse BB10. The 49th residueof Vx was demonstrated to play a crucial role in the antigenantibodyinteraction by 3D structure analysis with a computermodeling system. 相似文献
5.
Brunger Axel T.; Clore G.Marius; Gronenborn Angela M.; Karplus Martin 《Protein engineering, design & selection : PEDS》1987,1(5):399-406
A series of three-dimensional structures of the 129 fragmentof human growth hormone releasing factor in trifluoroethanolhave been determined by molecular dynamics and distance geometrymethods. The resulting structures satisfy information from nuclearOverhauser effect (NOE) distance data and an empirical potentialenergy function. Although the polypeptide was found to havean ordered structure in all simulations, the NOE data were notsufficient for global convergence to a unique three-dimensionalgeometry. Several satisfactory structures have been determined,all of which are extended conformations consisting of a shortß-strand and two -helices (residues 613 andresidues 1629) connected by short segments of less welldefined secondary structure. Because of the lack of NOE dataconnecting the helix segments, their relative orientation isnot uniquely determined. 相似文献
6.
In an attempt to facilitate crystallization, engineered cysteineswere used to promote formation of a backtobackdimer that occurs in different crystal forms of wildtypeand mutant T4 lysozymes. The designed double mutant, N68C/A93C,in which the surface residues Asn68 and Ala93 were replacedby cysteines, formed dimers in solution and crystallized isomorphouslyto wildtype, but at a much faster rate. Overall, themutant structure remained very similar to wildtype despite theformation of two intermolecular disulfide bridges. The crystalsof crosslinked dimers had thermal factors somewhat lowerthan wildtype, indicating reduced mobility, but did notdiffract to noticeably higher resolution. Introduction of thesame cross-links was also used to obtain crystals in a differentspace group of a T4 lysozyme mutant that could not be crystallizedpreviously. The results suggest that the formation of the lysozymedimer is a critical intermediate in the formation of more thanone crystal form and that covalent crossUnking of theintermediate accelerates nucleation and facilitates crystalgrowth. The disulfide crosslinks are located on the backof the molecule and formation of the crosslinked dimerappears to leave the active sites completely unobstructed. Nevertheless,the crosslinked dimer is completely inactive. One explanationfor this behavior is that the disulfide bridges prevent hinge-bendingmotion that may be required for catalysis. Another possibilityis that the formation of the dimer increases the overall bulkof the enzyme and prevents its access to the susceptible glycosidkbonds within the cell wall substrate 相似文献
7.
Molecular dynamics simulation of winter flounder antifreeze protein variants in solution: correlation between side chain spacing and ice lattice 总被引:1,自引:0,他引:1
Jorgensen H.; Mori M.; Matsui H.; Kanaoka M.; Yanagi H.; Yabusaki Y.; Kikuzono Y. 《Protein engineering, design & selection : PEDS》1993,6(1):19-27
The solution structure of the 38 amino acid C-terminal regionof the precursor for the HPLC-6 antifreeze protein from winterflounder has been investigated with molecular dynamics usingthe AMBER software. The simulation for the peptide in aqueoussolution was carried out at a constant temperature of 0°Cand at atmospheric pressure. The simulation covered 120 ps andthe results were analyzed based on data sampled upon reachinga stable equilibrium phase. Information has been obtained onthe quality of constant temperature and pressure simulations,the solution structure and dynamics, the hydrogen bonding network,the helix-stabilizing role of terminal charges and the interactionwith the surrounding water molecules. The Lys18Glu22interactions and the terminal charged residues are found tostabilize a helical structure with the side chains of Thr2,Thr13, Thr24 and Thr35 equally spaced on one side of the helix.The spacing between oxygen atoms in the hydroxyl group of thethreonine side chains exhibits fluctuations of the order of23 Å during the 120 ps of simulation, but valuessimultaneously close to the repeat distance of 16.6 Åbetween oxygen atoms along the [0112] direction in ice are observed.Furthermore, two engineered variants were studied using thesame simulation protocol. 相似文献
8.
Peelman Frank; Vanloo Berlinda; Perez-Mendez Oscar; Decout Anne; Verschelde Jean-Luc; Labeur Christine; Vinaimont Nicole; Verhee Annick; Duverger Nicolas; Brasseur Robert; Vandekerckhove Joel; Tavernier Jan; Rosseneu Maryvonne 《Protein engineering, design & selection : PEDS》1999,12(1):71-78
Lecithin cholesterol acyltransferase (LCAT) is an interfacialenzyme active on both high-density (HDL) and low-density lipoproteins(LDL). Threading alignments of LCAT with lipases suggest thatresidues 5074 form an interfacial recognition site andthis hypothesis was tested by site-directed mutagenesis. The(5668) deletion mutant had no activity on any substrate.Substitution of W61 with F, Y, L or G suggested that an aromaticresidue is required for full enzymatic activity. The activityof the W61F and W61Y mutants was retained on HDL but decreasedon LDL, possibly owing to impaired accessibility to the LDLlipid substrate. The decreased activity of the single R52A andK53A mutants on HDL and LDL and the severer effect of the doublemutation suggested that these conserved residues contributeto the folding of the LCAT lid. The membrane-destabilizing propertiesof the LCAT 5668 helical segment were demonstrated usingthe corresponding synthetic peptide. An M65NN66M substitutiondecreased both the fusogenic properties of the peptide and theactivity of the mutant enzyme on all substrates. These resultssuggest that the putative interfacial recognition domain ofLCAT plays an important role in regulating the interaction ofthe enzyme with its organized lipoprotein substrates. 相似文献
9.
Intrahelical side chainside chain (scsc) interactionsare assumed to play a crucial role in the formation and stabilityof -helices, yet it was found that only 37.2% of all helicalresidues are involved in such close contacts, assuming a specificminimum contact distance. The majority (58.0%) of these weredetected between residues with amino acid sequence spacing i,i + 4. The low frequency of intrahelical scsc contactswith sequence separations i, i + 1 and ii, i + 3, each observedwith only about one-third of the i, i + 4 counts, can be directlyand generally attributed to the absence of the g- conformationin helices for the dihedral angle X1- However, if it was assumedthat each side chain may maximally make only one scsccontact, as most commonly observed, the percentage of contactingpairs increased relative to the maximum possible pairs for agiven sequence spacing by a factor of {small tilde}4, e.g. from20.9 to 81.7% for i, i + 4 contacts. Stereochemical reasonsare also given for the observation that i, i + 3 contacts arecomposed largely of ion or polar pairs, while hydrophobic residuesdominate the i, i + 4 contacts. No significantly increased densityof intrahelical scsc contacts with increasing helix lengthwas found. Although there were generally fewer intrahelicalcontacts between buried helical residues when more contactswere made to the tertiary protein environment, the number ofintrahelical contacts did not increase with increasing solventexposure of the helices. Implications for helix design and thepacking of helices are discussed. 相似文献
10.
Yilmaz Selma; Widersten Mikael; Emahazion Tesfai 《Protein engineering, design & selection : PEDS》1995,8(11):1163-1169
Two mutant forms of human glutathione transferase (GST) Al1with affinity for metal ions were constructed by introductionof His residues by sitedirected mutagenesis. A mutant,2His, contained the mutations Lys84Gln, Asp85His andGlu88His, and another, 5His, contained the mutationsTyr79His, Asn80His, Lys84His, Asp85His and Glu88HLs. The mutantproteins were obtained in good yields (40150 mg per 3I culture) by heterologous expression in Escherichia coli. Themutant enzymes possessed novel binding affinities for Ni(II)and Zn(II) ions, as demonstrated by immobilized metal ion affinitychromatography. The mutant with two novel His residues (2Hismutant) did not bind as tightly to immobilized Nifll) as didthe mutant with five novel His residues (5His mutant).When tested for affinity to immobilized Zn(II), only the 5Hismutant remained bound to the column. The affinity of the 5Hismutant for Ni(II) ions in solution was determined by bindingexperiments in an aqueous polymeric twophase system.Analysis of the binding curve showed two binding sites per enzymesubunit and a dissociation constant of 6.7 1 . 6 M. The kineticconstants kcat, Km and kcat/km for the reaction with glutathioneand lchloro2,4dinitrobenzene were determinedby steadystate kinetic analysis and the parameter valuesfor the mutant forms were found to be indistinguishable fromthose obtained for the wildtype GST Al1. The differencesin surface charge in the mutant proteins as compared with thewildtype enzyme did not alter the pH dependence of kcat.The results provide an alternative method for purification offully active recombinant GST Al1 by the introductionof novel metal binding sites. The data also showed that twoHis residues are sufficient for Ni(II) binding. 相似文献
11.
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
12.
Tseitin Vladimir M.; Nikiforovich Gregory V. 《Protein engineering, design & selection : PEDS》1999,12(4):305-311
A computational procedure for predicting the arrangement ofan isolated helical fragment across a membrane was developed.The procedure places the transmembrane helical segment intoa model triple-phase system `wateroctanolwater';pulls the segment through the membrane, varying its `global'position as a rigid body; optimizes the intrahelical and solvationenergies in each global position by `local' coordinates (dihedralangles of side chains); and selects the lowest energy globalposition for the segment. The procedure was applied to 45 transmembranehelices from the photosynthetic reaction center from Rhodopseudomonasviridis, cytochrome c oxidase from Paracoccus denitrificansand bacteriorhodopsin. In two thirds of the helical fragmentsconsidered, the procedure has predicted the vertical shiftsof the fragments across the membrane with an accuracy of 0.15± 3.12 residues compared with the experimental data.The accuracy for the remaining 15 fragments was 2.17 ±3.07 residues, which is about half of a helix turn. The procedurepredicts the actual membrane boundaries of transmembrane helicalfragments with greater accuracy than existing statistical methods.At the same time, the procedure overestimates the tilt valuesfor the helical fragments. 相似文献
13.
Probing the nature of substrate binding in Humicola lanuginosa lipase through X-ray crystallography and intuitive modelling 总被引:1,自引:0,他引:1
Lawson David M.; Brzozowski Andrzej M.; Rety Stephane; Verma Chandra; Dodson G. Guy 《Protein engineering, design & selection : PEDS》1994,7(4):543-550
The catalytic triad of the neutral lipase from Humicola lanuginosais buried by a short helix under aqueous conditions renderingthe enzyme inactive. Upon adsorption to a lipid substrate interfacethis helix is displaced, thereby exposing the active site (interfacialactivation). By covalently linking inhibitors to the activeserine, it is possible to crystallize the enzyme in an interfaciallyactivated state. Two such structures are reported here whichmimic the tetrahedral transition states of lipolysis. To date,no crystal structures of a lipasetriglyceride complexexist for this enzyme. Therefore, possible interactions betweenthis lipase and its substrate have been analysed through molecularmodelling. 相似文献
14.
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
15.
Effects of amino acid substitutions in the hydrophobic core of {alpha}-lactalbumin on the stability of the molten globule state 总被引:1,自引:0,他引:1
Uchiyama Hidefumi; Perez-Prat Eva M.; Watanabe Kimitsuna; Kumagai Izumi; Kuwajima Kunihiro 《Protein engineering, design & selection : PEDS》1995,8(11):1153-1161
Five mutant lactalbumins, with one or two amino acidsubstitution(s) in the B helix, were engineered to examine therelation between the stability of the molten globule state andthe hydrophobicity of these amino acids. The mutation sites(Thr29, Ala30 and Thr33) have been chosen on the basis of comparisonof the amino acid sequences of goat, bovine and gunea pig lactalbumin,in which the guinea pig protein shows a remarkably more stablemolten globule than the other proteins. The recombinant proteinswere expressed Escherichia coli and then purified and refoldedefficiently to produce the active proteins. The stability ofthe molten globule state of these engineered proteins has beeninvestigated by ureainduced unfolding transition underan acidic condition (pH 2.0), where the molten globule stateis stable in the absence of urea. The results show that themolten globule state is stabilized by the amino acid substitutionswhich raise the hydrophobicity of the residues, suggesting thatthe hydrophobic core in a globular protein plays an importantrole in the stability of the molten globule state. The changein stabilization free energy of the molten globule state causedby each amino acid substitution has been evaluated, and molecularmechanisms of stabilization of the molten globule state arediscussed. 相似文献
16.
Clore G.Marius; Gronenborn Angela M.; Kjaer Mogens; Poulsen Flemming M. 《Protein engineering, design & selection : PEDS》1987,1(4):305-311
The solution structure of the 64 residue structured domain (residues2083) of barley serine proteinase inhibitor 2 (BSPI-2)is determined on the basis of 403 interproton distance, 34 øbackbone torsion angle and 26 hydrogen bonding restraints derivedfrom n.m.r. measurements. A total of 11 converged structureswere computed using a metric matrix distance geometry algorithmand refined by restrained molecular dynamics. The average rmsdifference between the final 11 structures and the mean structureobtained by averaging their coordinates is 1.4±0.2 Åfor the backbone atoms and 2.1±0.1 A for all atoms. Theoverall structure, which is almost identical to that found byX-ray crystallography, is disc shaped and consists of a centralfour component mixed parallel and antiparallel ß-sheetflanked by a 13 residue helix on one side and the reactivesite loop on the other. 相似文献
17.
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The structures of the interfaces of nine dimeric and nine tetramericproteins have been analyzed and have been seen to follow generalprinciples. These interfaces are combinations of four structuralmotifs, which resemble features of monomeric proteins. Theseare: (i) extended beta sheet; (ii) helixhelix packing;(iii) sheetsheet packing; and (iv) loop interactions.Other common structural features in the interfaces studied aretwo-fold symmetry, charged hydrogen bonds and channel formation(found only in tetramers). Monomermonomer interfacesare intermediate in hydrophobicity and charge between the interfacesbetween secondary structures of monomeric proteins and the exteriorsof monomeric proteins. A typical interface has one of the firstthree of the structural motifs at its centre and loop interactionsaround the outside, where most of the charge resides. 相似文献
19.
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
20.
Allemann Rudolf K.; Presnell Scott R.; Benner Steven A. 《Protein engineering, design & selection : PEDS》1991,4(7):831-835
A comparison of the sequences of three homologous ribonucleases(RNase A, angiogenin and bovine seminal RNase) identifies threesurface loops that are highly variable between the three proteins.Two hypotheses were contrasted: (i) that this variation mightbe responsible for the different catalytic activities of thethree proteins; and (ii) that this variation is simply an exampleof surface loops undergoing rapid neutral divergence in sequence.Three hybrids of angiogenin and bovine pancreatic ribonuclease(RNase) A were prepared where regions in these loops taken fromangiogenin were inserted into RNase A. Two of the three hybridshad unremarkable catalytic properties. However, the RNase Amutant containing residues 6374 of angiogenin had greatlydiminished catalytic activity against uridylyl-(3' 5')-adenosine(UpA), and slightly increased catalytic activity as an inhibitorof translation in vitro. Both catalytic behaviors are characteristicof angiogenin. This is one of the first examples of an engineeredexternal loop in a protein. Further, these results are complementaryto those recently obtained from the complementary experiment,where residues 5970 of RNase were inserted into angiogenin[Harper and Vallee (1989) Biochemistry, 28, 18751884].Thus, the external loop in residues 6374 of RNase A appearsto behave, at least in part, as an interchangeable modulethat influences substrate specificity in an enzyme in a waythat is isolated from the influences of other regions in theprotein. 相似文献