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1.
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
2.
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
3.
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
4.
Searle Mark S.; Zerella Rosa; Williams Dudley H.; Packman Leonard C. 《Protein engineering, design & selection : PEDS》1996,9(7):559-565
The conformational properties of protein fragments have beenwidely studied as models of the earliest initiation events inprotein folding. While native-like -helices and ß-turnshave been identified, less is known about the factors that underlyß-sheet formation, in particular ß-hairpins,where considerably greater long-range order is required. TheN-terminal 20 residue sequence of native ferredoxin I (fromthe blue-green alga Aphanothece sacrum ) forms a ß-hairpinin the native structure and has been studied in isolation byNMR and CD spectroscopy. Local native-like interactions aloneare unable to stabilize significantly a folded conformationof the 20-residue fragment in purely aqueous solution. However,we show that the addition of low levels of organic co-solventspromotes formation of native-like ß-hairpin structure.The results suggest an intrinsic propensity of the peptide toform a native-like ß-hairpin structure, and that theorganic co-solvent acts in lieu of the stabilizing influenceof tertiary interactions (probably hydrophobic contacts) whichoccur in the folding of the complete ferredoxin sequence. Thestructure of the isolated hairpin, including the native-likeregister of interstrand hydrogen bonding interactions, appearsto be determined entirely by the amino acid sequence. The solventconditions employed have enabled this intrinsic property tobe established. 相似文献
5.
White Christopher E.; Hunter Michael J.; Meininger David P.; White Lisa R.; Komives Elizabeth A. 《Protein engineering, design & selection : PEDS》1995,8(11):1177-1187
Fragments of human thrombomodulin (TM) have been expressed inlarge quantities in the Pichia pastoris yeast expression systemand purified to homogeneity. Fermentation of P.pastoris resultedin yields of 170 mg/1 TM. Purification to homogeneity resultedin an overall 10% yield, so that quantities of 20 mgpurified fragments can be readily obtained. Smaller fragmentsof TM, such as the individual fourth or fifth domains, werenot active, nor were equimolar mixtures of the two domains.These results demonstrate that the fourth and fifth epidermalgrowth factor (EGF)like domains together comprise thesmallest active fragment of TM. The fragment containing thefourth and fifth EGFlike domains (TMEGF(45)] had 10%the specific activity of rabbit TM. Comparison of the M388Lmutant TMEGF(45) fragment with the same mutant TMEGF(456)fragment showed that the fragment with the sixth domain hada 10fold better Km value for thrombin than the fragmentthat did not contain the sixth domain; this factor completelyaccounts for the higher specific activity of the fragments containingthe sixth domain. Comparison of the wildtype and M388Lmutants showed that the M388L mutation resulted in a 2foldincrease in kcat for the activation of protein C by the thrombinTMfragment complex, completely accounting for the 2foldincrease in specific activity of these mutant fragments. 相似文献
6.
Multimer formation as a consequence of separate homodimerization domains: the human c-Jun leucine zipper is a transplantable dimerization module 总被引:1,自引:0,他引:1
Riley L.G.; Ralston G.B.; Weiss A.S. 《Protein engineering, design & selection : PEDS》1996,9(2):223-230
Human c-Jun and c-Fos leucine zipper domains were examined fortheir ability to serve as autonomous dimerization domains aspart of a heterologous protein construct. Schistosoma japonicumglutathione S-transferase (GST) was fused to recombinant Junleucine zipper (rJunLZ) and Fos leucine zipper (rFosLZ) domains.SDSPAGE snapshot analyses based on disulphidelinkage of monomers demonstrated the ability of rJunLZ to functionas a dimerization motif in a foreign protein environment. Sterichindrance prevented formation of rJunLZGST::rFosLZGSTheterodimers whereas rJunLZGST::rFosLZ and rJunLZ::rFosLZGSTformed readily. Furthermore, rJunLZGST generated homodimerssuggesting fusion protein heterodimers interact differentlyto homodimers. Gel filtration chromatography confirmed thatGST is a dimer in solution and that attachment of a leucinezipper domain allows further interactions to take place. Sedimentationequilibrium analyses showed that GST is a stable dimer (Ka >106 M-1) with no higher multimeric forms. rFosLZGST weaklyassociates beyond a dimer (Ka {small tilde}4x105 M-1) and rJunLZGSTassociates indefinitely (Ka {small tilde}4x106 M-1), consistentwith an isodesmic model of association. The interaction of theseleucine zippers independently of GST association demonstratestheir utility in the modification of proteins when multimerformation is desired. 相似文献
7.
The cDNA encoding the human trifunctional enzyme methylenetetrahydrofolatedehydrogenase-methenyltetrahydrofolate cyclohydrolase-formyltetrahydrofolatesynthetase was engineered to contain a prokaryotic ribosomebinding site and was expressed under the bacteriophage T7 RNApolymerase promoter in Escherichia coli. Site-directed mutagenesiswas used to prepare constructs that encode separately the dehydrogenase/cyclohydrolase(D/C) domain as amino acid residues 1301, and the synthetase(Syn) domain as residues 304935. Both domains formedactive enzymes thereby demonstrating their ability to fold independently.The full-length enzyme, D/C and Syn domains were expressed atlevels 4-, 55- and 3-fold higher than the specific activitiesfound in liver. Additional mutagenesis and independent expressionof domains further defined the interdomain region to includeamino acids 292310. The D/C domain was purified to homogeneityby a single affinity chromatographic step, and the full-lengthprotein in a twostep procedure. The kinetic properties of theD/C domain appear unaltered from those of the trifunctionalenzyme. 相似文献
8.
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
9.
Stabilization of lysozyme against irreversible inactivation by alterations of the Asp-Gly sequences 总被引:3,自引:0,他引:3
Tomizawa Hideyuki; Yamada Hidenori; Hashimoto Yoshio; Imoto Taiji 《Protein engineering, design & selection : PEDS》1995,8(10):1023-1028
Site-directed mutagenesis was performed at Asp-Gly (4849,6667, 101102) and Asn-Gly (103104) sequencesof hen egg-white lysozyme to protect the enzyme against irreversiblethermoinactivation. Because the lysozyme inactivation was causedby the accumulation of multiple chemical reactions, includingthe isomerization of the Asp-Gly sequence and the deamidationof Asn [Tomizawa et al.(1994) Biochemistry, 33, 1303213037],the suppression of these reactions by the substitution of Glyto Ala, or the introduction of a sequence of human-type lysozyme,was attempted and the mutants (where each or all labile sequenceswere replaced) were prepared. The substitution resulted in thereversible destabilization from 1 to 2 kcal/mol per substitution.The destabilization was caused by the introduction of ß-carbonto the constrained position that had conformational angles withinthe allowed range for the Gly residue. Despite the decreasein the reversible conformational stability, the mutants hadmore resistance to irreversible inactivation at pH 4 and 100°C.In particular, the rate of irreversible inactivation of themutant, which was replaced at four chemically labile sequences,was the latest and corresponded to 18 kcal/mol of the reversibleconformational stability. Therefore, replacement of the chemicallylabile sequence was found to be more effective at protectingenzymes against irreversible thermoinactivation than at strengtheningreversible conformational stability. 相似文献
10.
McDonald Shawn M.; Willson Richard C.; McCammon J.Andrew 《Protein engineering, design & selection : PEDS》1995,8(9):915-924
The titration behavior of the ionizable residues of the HyHEL-5henegg lysozyme complex and its individual components has beenstudied using continuum electrostatic calculations. Severalresidues of HyHEL-5 had pKa values shifted away from model valuesfor isolated residues by more than three pH units. Shifts awayfrom the model values were smaller for the residues of hen egglysozyme. A moderate variation in the pKa values of the titratablegroups was observed upon increase of the ionic strength from0 to 100 mM, amounting to 12 pH units in most cases.Under physiological conditions, the net charge of HyHEL-5 wasopposite that for hen egg lysozyme. Several residues, includingthose involved in the ArgGlu salt bridges that have beenproposed to be important in antibody-antigen binding, had pKavalues that were changed significantly upon binding. The maintitration event upon antibody-antigen binding appears to beloss of a proton from residue GluH50 of the Fv molecule. Thelimitations of our calculation methods and the role they mightplay in the design of antibodies for use in assays, sensorsand separations are discussed 相似文献
11.
Eidsness Marly K.; O'Dell Sonia E.; Kurtz Donald M. Jr; Robson Robert L.; Scott Robert A. 《Protein engineering, design & selection : PEDS》1992,5(4):367-371
A synthetic gene based on the published amino acid sequencefor Clostridium pasteurianum rubredoxin was constructed, clonedin Escherichia coli 71/18 and expressed using the T7 RNA polymerase/promotersystem in E.coli HMS273. UV/visible spectroseopy and metal analysesindicated that the asisolated synthetic gene productis a mixture of holo(i.e. ironcontaining) rubredoxinand zincsubstituted rubredoxin, with the latter amountingto {small tilde} 70% of the total rubredoxin. Hie UV/visibleabsorption and resonance Raman spectra of the cloned holorubredoxinare characteristic of the native rubredoxintype ironsite. Nterminal amino acid sequencing suggests that thegene product consists of at least three polypeptide specieswith the initial sequences (approximate relative abundances):MetMetLys... (63%), blocked (30%) and MetLys...(7%). The blocked portion presumably consists of a mixture ofnMetMetLys... and nMet-Lys..., wherenMet represents an aminoblocked methionine residue. 相似文献
12.
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
13.
Frankel Arthur; Tagge Edward; Chandler John; Burbage Chris; Willingham Mark 《Protein engineering, design & selection : PEDS》1996,9(4):371-379
Three distinct double-site and two single-site ricin B chain(RTB) mutants were expressed in Spodoptera frugiperda insectcells and purified from infected cell supernatants. The yieldsof recombinant proteins were 0.010.2 mg/1. The purityafter monoclonal antibody affinity chromatography was 120%.The mutant proteins were soluble, immuno-reactive with monoclonalantibodies and polyclonal antibodies to RTB and demonstratedmolecular weights of 32 kDa, similar to plant RTB. All threedouble-site and both single-site mutants bound asialofetuinand mammalian cell surfaces based on an asialofetuin ELISA andcell binding immunofluorescence assay. While one double-sitemutant, W37S/Y248S, had a 1 log drop in sugar binding, the othertwo double-site mutants W37S/Y248H and D22E/D234E had 2 logreductions in sugar binding. Each mutant reassociated efficiently(2575%) with plant ricin A chain (RTA) to form cytotoxicheterodimers. The concentration of protein required to reduceprotein synthesis 50% (ID50) was 1 log higher than plant ricinfor W37S/Y248S-RTA and the single-site mutant heterodimers,Q35N-RTA and D22E-RTA and 2 logs higher than plant ricin forthe other two double-site mutant heterodimers. The results suggestamino acid residues in both the 1 and 2 subdomains of RTB participatein sugar binding. However, other subdomains must contributeto the avidity of ricin for cell surface oligosaccharides. 相似文献
14.
Genetic algorithms are very efficient search mechanisms whichmutate, recombine and select amongst tentative solutions toa problem until a near optimal one is achieved. We introducethem as a new tool to study proteins. The identification andmotivation for different fitness functions is discussed. Theevolution of the zinc finger sequence motif from a random startis modelled. User specified changes of the repressor structurewere simulated and critical sites and exchanges for mutagenesisidentified. Vast conformational spaces are efficiently searchedas illustrated by the ab initio folding of a model protein ofa four ß strand bundle. The genetic algorithm simulationwhich mimicked important folding constraints as overall hydrophobicpackaging and a propensity of the betaphilic residues for transpositions achieved a unique fold. Cooperativity in the ßstrand regions and a length of 35 for the interconnectingloops was critical. Specific interaction sites were considerablyless effective in driving the fold. 相似文献
15.
Chiche L.; Heitz A.; Padilla A.; Le-Nguyen D.; Castro B. 《Protein engineering, design & selection : PEDS》1993,6(7):675-682
The trypsin carboxypeptidase peptide inhibitor (TCPI) whichinhibits both trypsin and carboxypeptidase A has been chemicallyengineered by modification of the Ecballium elaterium trypsininhibitor II (EETI-II). The solution conformation of TCPI, studiedby two-dimensional nuclear magnetic resonance, was shown tobe very close to those of squash inhibitors. Only limited deviationsof the trypsin binding loop compared to its location in theEETI-II/trypsin complex were detected. It was also shown thatthe position of the C-terminal tail did not significantly changefrom the position observed in the complex between carboxypeptidaseA and the potato carboxypeptidase inhibitor (PCI). The conformationof TCPI was carefully compared with the PCI one and a new structuralalignment between the two microproteins is proposed. This alignmentpoints out the very good conservation in the two inhibitorsof a subdomain comprising segments 715, 1922 and2528. Most importantly, the 219 disulfide bridgeof TCPI was not structurally conserved in PCI and appeared tobe rather unimportant for the early folding process of thesemolecules. This result agrees with the recent observation thatthe 219 bridge is the last to be formed in the foldingof the squash inhibitor EETI-II and suggests that this is alsothe case during the folding of the potato carboxypeptidase inhibitor. 相似文献
16.
Effect of amino acid deletions in the O-glycosylated region of Aspergillus awamori glucoamylase 总被引:1,自引:0,他引:1
Libby Carol Baker; Cornett Catherine A.G.; Reilly Peter J.; Ford Clark 《Protein engineering, design & selection : PEDS》1994,7(9):1109-1114
Aspergillus awamori glucoamylase (GA) contains globular catalyticand starch-binding domains (residues 1471 and 509616,respectively). A heavily O-glycosylated sequence comprises twoparts. The first (residues 441471) in the crystal structurewraps around an /-barrel formed by residues 1440. Thesecond (residues 472508) is an extended, semi-rigid linkerbetween the two domains. To investigate the functional roleof this linker, we made internal deletions to remove residues466512 (GA1), 485512 (GA2) and 466483 (GA3).GA2 and GA3 were expressed in Saccharomyces cerevisiae culturesupernatants at 60 and 20% the wild-type level, respectively,while GA1 was almost undetectable. Western blots comparing extracellularand intracellular fractions indicated that the region deletedin GA3 was critical for secretion, while the region deletedin GA2 contributed to the production of a stable enzyme structure.The activities of purified GA2 and GA3 on soluble and insolublestarch were similar to those of wild-type GA, indicating thatfor soluble starch their deletions did not affect the catalyticdomain and for insoluble starch the linker does not coordinatethe activities of the catalytic and starch-binding domains.The deletions had a significant negative effect on GA2 and GA3thermos tabilities. 相似文献
17.
Doi T.; Tokunaga T.; Ohtsuka E.; Hiraki Y.; Suzuki F.; Ikehara M. 《Protein engineering, design & selection : PEDS》1986,1(1):17-21
We have constructed three different plasmids containing partsof the human growth hormone gene using chemically synthesizedoligomers and cloned them for the purpose of expressing themin Escherichia coli. AB, B and BC gene segments correspondingto ABhGH (residue 1138), BhGH (residue 44138)and BChGH (residue 44192) were placed under the controlof a tryptophan promoter in the expression vector. Upon inductionwith 3-indolylacrylic acid, ABhGH accumulated in cells but theBhGH and BChGH segments were not detected appreciably. Northernblotting analysis showed that the amount of mRNA transcribedfrom the AB gene segment was about ten-fold higher than thatfrom the B or BC gene segment. ABhGH was found to have insulin-likegrowth factor I (IGF-I) activity, which could be explained bythe hydrophilicity curves of these proteins. 相似文献
18.
Gore Michael G.; Ferris William F.; Popplewell Andrew G.; Scawen Michael; Atkinson Tony 《Protein engineering, design & selection : PEDS》1992,5(6):577-582
A fusion protein, consisting of the N-terminal 81 amino acidsfrom an inactive bovine DNase I (Q38,E39E38,Q39) andtwo sequential synthetic IgG-binding domains based upon domainB of Protein A from Staphylococcus aureus has been shown tobind to porcine IgG with a similar affinity and pH profile toProtein A. The same residue in each B domain (Tyr111 and Tyr169)has been mutated by cassette mutagenesis to Ser, Glu, His, Lysor Arg and the effect of the mutation on binding interactionswith porcine IgG investigated. The evidence presented suggeststhat the interactions at the B domain are highly sensitive tothe presence of a charged residue. 相似文献
19.
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
20.
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