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1.
Improvement of thermal stability of Streptomyces cholesterol oxidase by random mutagenesis and a structural interpretation 总被引:1,自引:0,他引:1
Nishiya Y; Harada N; Teshima SI; Yamashita M; Fujii I; Hirayama N; Murooka Y 《Protein engineering, design & selection : PEDS》1997,10(3):231-235
Random mutagenesis was used to enhance the thermal stability of
Streptomyces cholesterol oxidase. Four thermostable mutants were isolated
and the following amino acid substitutions were identified: Ser103 to Thr
(mutant S103T), Val121 to Ala (mutant V121A), Arg135 to His (mutant R135H)
and Val145 to Glu (mutant V145E). The wild-type and mutant enzymes were
purified and characterized. The properties of mutants S103T, V121A and
R135H were similar to those of the wild type but they showed improved
thermal stability. When the V145E mutation was introduced, the thermal
stability of the enzyme was markedly increased and the optimum pH was
desirably changed to encompass a broad range from acid to alkali. Analysis
of multiple mutants constructed by site- directed mutagenesis showed that
all the mutations except that of R135H had an additive influence on the
other mutations. These mutational effects are discussed in terms of a
three-dimensional structural model of the enzyme constructed on the basis
of homology modelling.
相似文献
2.
Japrung D Chusacultanachai S Yuvaniyama J Wilairat P Yuthavong Y 《Protein engineering, design & selection : PEDS》2005,18(10):457-464
Sufficient solubility of the active protein in aqueous solution is a prerequisite for crystallization and other structural studies of proteins. In this study, we have developed a simple and effective in vivo screening system to select for functionally active proteins with increased solubility by using Plasmodium falciparum dihydrofolate reductase (pfDHFR), a well-known malarial drug target, as a model. Prior to the dual selection process, pfDHFR was fused to green fluorescent protein (GFP), which served as a reporter for solubility. The fusion gene was used as a template for construction of mutated DNA libraries of pfDHFR. Two amino acids with large hydrophobic side chains (Y35 and F37) located on the surface of pfDHFR were selected for site-specific mutagenesis. Additionally, the entire pfDHFR gene was randomly mutated using error-prone PCR. During the first step of the dual selection, mutants with functionally active pfDHFR were selected from two libraries by using bacterial complementation assay. Fluorescence signals of active mutants were subsequently measured and five mutants with increased GFP signal, namely Y35Q + F37R, Y35L + F37T, Y35G + F37L and Y35L + F37R from the site-specific mutant library and K27E from the random mutant library, were recovered. The mutants were expressed, purified and characterized as monofunctional pfDHFR following excision of GFP. Our studies indicated that all mutant pfDHFRs exhibited kinetic properties similar to that of the wild-type protein. For comparison of protein solubility, the maximum concentrations of mutant enzymes prior to aggregation were determined. All mutants selected in this study exhibited 3- to 6-fold increases in protein solubility compared with the wild-type protein, which readily aggregated at 2 mg/ml. The dual selection system we have developed should be useful for engineering functionally active protein mutants with sufficient solubility for functional/structural studies and other applications. 相似文献
3.
Firefly luciferase is widely used in a number of areas of biotechnology and molecular biology. However, rapid inactivation of wild-type (WT) luciferases at elevated temperatures often hampers their application. A simple non-lethal in vivo screening scheme was used to identify thermostable mutants of luciferase in Escherichia coli colonies. This scheme allowed carrying out each cycle of mutagenesis in a rapid and efficient manner. Four rounds of directed evolution were conducted on a part of the gene coding for amino acid residues 130-390 of Luciola mingrelica luciferase. The resultant mutant designated 4TS had a half-life of 10 h at 42°C, which is 65-fold higher compared with the WT luciferase. Moreover, the mutant 4TS showed a 1.9-fold increase in specific activity, 5.7-fold reduction of K(m) for ATP and a higher-temperature optimum compared with the WT enzyme. 4TS contains eight mutations, four?of which are suggested to be mainly responsible for the enhancement of thermostability: R211L, A217V, E356K and S364C. Thus, directed evolution with non-lethal colony screening for in vivo bioluminescence activity proved to be an effective and efficient approach for increasing thermal stability of luciferase while retaining high catalytic activity. 相似文献
4.
Hydrophobicity engineering of cholera toxin A1 subunit in the strong adjuvant fusion protein CTA1-DD
Agren Lena; Norin Martin; Lycke Nils; Lowenadler Bjorn 《Protein engineering, design & selection : PEDS》1999,12(2):173-178
Protein engineering of the cholera toxin A1 subunit (CTA1) fusedto a dimer of the Ig-binding D-region of Staphylococcus aureusprotein A (DD) was employed to investigate the effect of specificamino acid changes on solubility, stability, enzymatic activityand capacity to act as an adjuvant in vivo. A series of CTA1-DDanalogues were selected by a rational modeling approach, inwhich surface-exposed hydrophobic amino acids of CTA1 were exchangedfor hydrophilic counterparts modeled for best structural fit.Of six different mutants initially produced, two analogues,CTA1Phe132Ser-DD and CTA1Pro185Gln-DD, were demonstrated tohave 50 and 70% increased solubility, respectively, at neutralpH. The double mutant CTA1Phe132Ser/Pro185Gln-DD was at leastthreefold more soluble, demonstrating an additive effect ofthe two mutations. Only the Phe132Ser analogue retained fullbiological activity and stability compared with the native CTA1-DDfusion protein. Two mutants, Pro185Gln and Phe31His mutations,exhibited unaltered ADP-ribosyltransferase activity in vitro,but demonstrated markedly reduced adjuvant function. Since thePro185 and Phe31 amino acids are located in close vicinity onthe distal side of the molecule relative to the enzymaticallyactive cleft, it is conceivable that this region is involvedin mediating a biological function, separate from the enzymaticactivity but intrinsic to the adjuvant activity of CTA1. 相似文献
5.
Nieba L; Honegger A; Krebber C; Pluckthun A 《Protein engineering, design & selection : PEDS》1997,10(4):435-444
By constructing Fv and single-chain Fv (scFv) fragments of antibodies, the
variable domains are taken out of their natural context in the Fab
fragment, where they are associated with the constant domains of the light
(CL) and heavy chain (CH1). As a consequence, all residues of the former
variable/constant domain interface become solvent exposed. In an analysis
of 30 non-redundant Fab structures it was found that at the former
variable/constant domain interface of the Fv fragment the frequency of
exposed hydrophobic residues is much higher than in the rest of the Fv
fragment surface. We investigated the importance of these residues for
different properties such as folding in vivo and in vitro, thermodynamic
stability, solubility of the native protein and antigen affinity. The
experimental model system was the scFv fragment of the anti-fluorescein
antibody 4-4-20, of which only 2% is native when expressed in the periplasm
of Escherichia coli. To improve its in vivo folding, a mutagenesis study of
three newly exposed interfacial residues in various combinations was
carried out. The replacement of one of the residues (V84D in VH) led to a
25-fold increase of the functional periplasmic expression yield of the scFv
fragment of the antibody 4-4-20. With the purified scFv fragment it was
shown that the thermodynamic stability and the antigen binding constant are
not influenced by these mutations, but the rate of the thermally induced
aggregation reaction is decreased. Only a minor effect on the solubility of
the native protein was observed, demonstrating that the mutations prevent
aggregation during folding and not of the native protein. Since the
construction of all scFv fragments leads to the exposure of these residues
at the former variable/constant domain interface, this strategy should be
generally applicable for improving the in vivo folding of scFv fragments
and, by analogy, also the in vivo folding of other engineered protein
domains.
相似文献
6.
Damnjanović J Takahashi R Suzuki A Nakano H Iwasaki Y 《Protein engineering, design & selection : PEDS》2012,25(8):415-424
Aimed to produce thermostable phosphatidylinositol (PI)-synthesizing phospholipase D (PLD), we initiated site-directed combinatorial mutagenesis followed by high-throughput screening. Previous site-directed combinatorial mutagenesis of wild-type Streptomyces PLD produced a mutant, DYR (W187D/Y191Y/Y385R) with PI-synthesizing ability. Deriving PI as a product of transphosphatidylation between phosphatidylcholine and myo-inositol, with myo-inositol in excess at high-temperature reaction conditions can increase yield due to enhanced solubility of this substrate. Thus, we improved DYR's thermostability by introduction of random mutations into selected amino acid positions having high B-factor. Screening of the libraries under restricted conditions yielded single-point mutants, specifically D40H, T291Y and R329G. Combinations of these point mutations yielded double (D40H/T291Y, D40H/R329G and T291Y/R329G) and triple (D40H/T291Y/R329G) mutants. PI synthesis at elevated temperatures pointed at D40H/T291Y as the most efficient enzyme. Circular dichroism analysis revealed D40H/T291Y to have increased melting temperature and postponed onset of thermal unfolding compared with DYR. Thermal tolerance study at 65°C confirmed D40H/T291Y's thermostability as its half-inactivation time was 8.7 min longer compared with DYR. This mutant had significantly less root-mean-square deviation change compared with DYR and showed no change in root-mean-square fluctuation when temperature shifts from 40 to 60°C, as determined by molecular dynamics analysis. Acquired different degrees of thermostability were also observed for several other DYR mutants. 相似文献
7.
Yao P; Xie Y; Wang YH; Sun YL; Huang ZX; Xiao GT; Wang SD 《Protein engineering, design & selection : PEDS》1997,10(5):575-581
Phenylalanine-35, which is a residue of the hydrophobic patch on the
surface of cytochrome b5, has been mutated into Tyr35, His35 and Leu35 to
elucidate the functions of the Phe35 and give further insight into the
roles of the hydrophobic patch and/or aromatic network. The effects of
these mutations on the heme environment, denaturation towards heating and
the denaturant urea, redox potential and stability of protein were studied.
The relative stability of cytochrome b5 and its mutants towards heating has
the order Phe35Tyr > wild type > Phe35Leu > Phe35His in the
oxidized state and wild type > Phe35Tyr > Phe35Leu > Phe35His in
the reduced state. All the mutants exhibit decreased reduction potentials:
Phe35Tyr -66 mV, Phe35His -51 mV and Phe35Leu -28 mV, which are more
negative than that of the wild type. The order of redox potential reflects
the relative stability in the oxidized and reduced states. A method of
producing multiple mutants at a single site of a gene is also described for
the first time.
相似文献
8.
Sierks Michael R.; Ford Clark; Reilly Peter J.; Svensson Birte 《Protein engineering, design & selection : PEDS》1989,2(8):621-625
Trpl20 of Aspergillus awamori glucoamylase has previously beenshown by chemical modification to be essential for activityand tentatively to be located near subsite 4 of the active site.To further test its role, restriction sites were inserted inthe cloned A.awamori gene around the Trpl20 coding region, andcassette mutagenesis was used to replace it with His, Leu, Pheand Tyr. All four mutants displayed 2% or less of the maximalactivity (kcat) of wild-type glucoamylase towards maltose andmaltoheptaose. MichaelLs constants (KM) of mutants decreased2- to 3-fold for maltose and were essentially unchanged formaltoheptaose compared with the wild type, except for a >3-fold decrease for maltoheptaose with the Trp120 Tyrmutant. This mutant also bound isomaltose more strongly andhad more selectivity for its hydrolysis than wild-type glucoamylase.A subsite map generated from malto-oligosaecharide substrateshaving 2 7 D-glucosyl residues indicated that subsites1 and 2 had greater affinity for D-glucosyl residues in theTrp120 Tyr mutant than in wild-type glucoamylase. Theseresults suggest that Trpl20 from a distant subsite is crucialfor the stabilization of the transition-state complex in subsites1 and 2. 相似文献
9.
目的对人内皮抑素(hES)基因进行定点突变,提高其在大肠杆菌中的可溶性表达量。方法根据Internet网站提供的关于hES的结构信息及其结构与功能方面的研究文献,设计突变位点;利用生物信息学的相关网站,对hES突变体进行结构预测,验证突变位点设计的合理性;采用重叠延伸PCR方法进行hES基因的定点突变,并将突变基因和未突变基因分别亚克隆至表达载体pGEX-4T-3,在大肠杆菌中进行融合表达,比较突变前后目的蛋白的可溶性表达量。结果三级结构预测结果表明突变位点设计合理,序列分析结果表明实现了hES基因的定点突变。突变基因的表达产物中,可溶性部分约占总表达量的40%,而未突变基因的表达产物几乎全部为包涵体。结论已成功对hES基因进行了定点突变,突变后的hES可溶性表达量得到了提高。 相似文献
10.
Teplyakov A.V.; van der Laan J.M.; Lammers A.A.; Kelders H.; Kalk K.H.; Misset O.; Mulleners L.J.S.M.; Dijkstra B.W. 《Protein engineering, design & selection : PEDS》1992,5(5):413-420
Serine endoproteases such as trypsins and subtilisins are knownto have an extended substrate binding region that interactswith residues P6 to P3' of a substrate. In order to investigatethe structural and functional effects of replacing residuesat the S4 substrate binding pocket, the serine protease fromthe alkalophilic Bacillus strain PB92, which shows homologywith the subtilisins, was mutated at positions 102 and 126128.Substitution of Val102 by Trp results in a 12fold increasein activity towards succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide(sAAPFpNA). An X-ray structure analysis of the V102W mutantshows that the Trp side chain occupies a hydrophobic pocketat the surface of the molecule leaving a narrow crevice forthe P4 residue of a substrate. Better binding of sAAPFpNA bythe mutant compared with the wild type protein as indicatedby the kinetic data might be due to the hydrophobic interactionof Ala P4 of the substrate with the introduced Trp102 side chain.The observed difference in binding of sAAPFpNA by protease PB92and thermitase, both of which possess a Trp at position 102,is probably related to the amino acid substitutions at positions105 and 126 (in the protease PB92 numbering).Kinetic data forthe variants obtained by random mutation of residues Serl26,Prol27 and Serl28 reveal that the activity towards sAAPFpNAincreases when a hydrophobic residue is introduced at position126. An X-ray diffraction analysis was carried out for the threeprotease PB92 mutants which have residues Serl26-Prol27-Serl28replaced by Met-Ala-Gly(MAG mutant), Phe-Gln-Ser(FQS mutant) and Asn-Ser-Ala (NSAmutant). Met 126 and Phel26 in the crystal structures of thecorresponding mutants are fixed in the same hydrophobic environmentas Trp102 in the V102W mutant.In contrast, Asnl26 in the NSAmutant is completely disordered in both crystal forms for whichthe structure has been determined. According to our kineticmeasurements none of the mutants with Met, Phe, Leu or Val atposition 126 binds sAAPFpNA better than the wild type enzyme.Resultsof the site-directed mutagenesis at position 127 imply thatpossible interaction of this residue with a substrate has almostno effect on activity towards sAAPFpNA and casein. 相似文献
11.
Hancock SM Corbett K Fordham-Skelton AP Gatehouse JA Davis BG 《Chembiochem : a European journal of chemical biology》2005,6(5):866-875
Two residues that have been implicated in determining the substrate specificity of the thermophilic beta-glycosidase from the archaeon Sulfolobus solfataricus (SsbetaG), a member of the glycosyl hydrolase family 1, have been mutated by site-directed mutagenesis so as to create more versatile catalysts for carbohydrate chemistry. The wild-type and mutated sequences were expressed in E. coli with a His(7)-tag to allow one-step chromatographic purification. The E432C and W433C mutations removed key interactions with the OH-4 and OH-3 of the sugar substrates, thus reducing the discrimination of glucose, galactose and fucose with respect to other glycosides. This resulted in two glycosidases with greatly broadened substrate specificities. Observed changes include a 24-fold increase in Man:Gal activity and an 18-fold increase in GalA:Gal activity. This promiscuous substrate tolerance was further illustrated by the parallel synthesis of a beta-glycoside library of glucose, galactose, xylose and mannose in one pot at 50 degrees C, in organic solvent. The synthetic potential of the catalysts was further evaluated through alkyl glycoside transglycosylation yields, including the first examples of synthesis of beta-mannosides and beta-xylosides with SsbetaG. 相似文献
12.
Béhar G Solé V Defontaine A Maillasson M Quéméner A Jacques Y Tellier C 《Protein engineering, design & selection : PEDS》2011,24(3):283-290
Directed evolution was used to generate IL-15 mutants with increased solubility and cytoplasmic over-expression in Escherichia coli. A protein solubility selection method was used in which the IL-15 gene was expressed as an N-terminal fusion to chloramphenicol acetyltransferase (CAT) as reporter protein. Clones that grew in the presence of high concentrations of chloramphenicol were then screened by ELISA to assay the binding activity of the IL-15-CAT fusion to the IL-15Rα Sushi domain. Two variants of IL-15, M38 and M253, containing five mutations and one mutation respectively, were selected with a dramatic improvement in solubility; the soluble concentration in cell culture was 12- to 18-fold higher, respectively, than for WT IL-15. Characterization of their binding to IL-15Rα and their ability to stimulate the T-cell growth response showed that M38 binds as strongly as native IL-15 to IL-15Rα and acts as an effective agonist of IL-15. 相似文献
13.
Improving Escherichia coli alkaline phosphatase efficacy by additional mutations inside and outside the catalytic pocket 总被引:1,自引:0,他引:1
Muller BH Lamoure C Le Du MH Cattolico L Lajeunesse E Lemaître F Pearson A Ducancel F Ménez A Boulain JC 《Chembiochem : a European journal of chemical biology》2001,2(7-8):517-523
We describe a strategy that allowed us to confer on a bacterial (E. coli) alkaline phosphatase (AP) the high catalytic activity of the mammalian enzyme while maintaining its high thermostability. First, we identified mutations, at positions other than those occupied by essential catalytic residues, which inactivate the bacterial enzyme without destroying its overall conformation. We transferred concomitantly into the bacterial enzyme four residues of the mammalian enzyme, two being in the catalytic pocket and two being outside. Second, the gene encoding the inactive mutant was submitted to random mutagenesis. Enzyme activity was restored upon the single mutation D330N, at a position that is 12 A away from the center of the catalytic pocket. Third, this mutation was combined with other mutations previously reported to increase AP activity slightly in the presence of magnesium. As a result, at pH 10.0 the phosphatase activity of both mutants D330N/D153H and D330N/D153G was 17-fold higher than that of the wild-type AP. Strikingly, although the two individual mutations D153H and D153G destabilize the enzyme, the double mutant D330N/D153G remained highly stable (T(m)=87 degrees C). Moreover, when combining the phosphatase and transferase activities, the catalytic activity of the mutant D330N/D153G increased 40-fold (k(cat)=3200 s-1) relative to that of the wild-type enzyme (k(cat)=80 s-1). Due to the simultaneous increase in K(m), the resulting k(cat)/K(m) value was only increased by a factor of two. Therefore, a single mutation occurring outside a catalytic pocket can dramatically control not only the activity of an enzyme, but also its thermostability. Preliminary crystallographic data of a covalent D330N/D153G enzyme-phosphate complex show that the phosphate group has significantly moved away from the catalytic pocket, relative to its position in the structure of another mutant previously reported. 相似文献
14.
Yonemoto W; McGlone ML; Grant B; Taylor SS 《Protein engineering, design & selection : PEDS》1997,10(8):915-925
When the catalytic (rC) subunit of cAMP-dependent protein kinase (cAPK) is
expressed in Escherichia coli, it is autophosphorylated at four sites,
Ser10, Ser139, Ser338 and Thr197 (49). Three of these sites, Ser10, Ser338
and Thr197, are also found in the mammalian enzyme. To understand the
functional importance of these phosphorylation sites, each was replaced
with Ala, Glu or Asp. The expression, solubility and phosphorylation state
of each mutant protein was characterized by immunoprecipitation following
in vivo labeling with 32Pi. When possible, isoforms were resolved and
kinetic properties were measured. The two stable phosphorylation sites in
the mammalian enzyme, Ser338 and Thr197, were shown to play different
roles. Ser338, which stabilizes a turn near the C-terminus, is important
for stability. Both rC(S338A) and rC(S338E) were very labile; however, the
kinetic properties of rC(S338E) were similar to the wild-type catalytic
subunit (C-subunit). Ser338 most likely helps to anchor the C-terminus to
the surface of the small lobe. Thr197 is in the activation loop near the
cleft interface. Mutagenesis of T197 caused a significant loss of catalytic
activity with increases in Kms for both peptide and MgATP, as well as a
small decrease in k(cat) indicating that this phosphate is important for
the correct orientation of catalytic residues at the active site.
Replacement of Ser139, positioned at the beginning of the E-helix, with Ala
had no effect on the kinetic parameters, stability or phosphorylation at
the remaining sites. In contrast, mutation of Ser10, located at the
beginning of the A-helix, produced mostly insoluble, inactive,
unphosphorylated protein, suggesting that this region, though far removed
from the active site, is structurally important at least for the expression
of soluble phosphoprotein in E.coli. Since the mutation of active site
residues as well as deletion mutants generate underphosphorylated proteins,
these phosphorylations in E.coli all result from autophosphorylation.
相似文献
15.
Kruse R.; Hengstenberg W.; Beneicke W.; Kalbitzer H.R. 《Protein engineering, design & selection : PEDS》1993,6(4):417-423
The phosphocarrier HPr (heat stable protein) of Staphylococcuscarnosus was modified by site-directed mutagenesis of the correspondingptsH gene in order to analyse the importance of amino acidswhich were supposed to be part of the active centre of the protein.Three residues which are conserved in all HPrs, Argl7, Prol8and Glu84, were mutated: Argl7 was changed to His (17RH) andPro18 and Glu84 were changed into Ala (18PA and 84EA). In addition,Leu86 was changed into Ala (86LA) and one mutant protein wasmissing the last six residues of the HPr (83). The wild typegene and all mutant genes were overexpressed and the gene productspurified to homogeneity. Three-dimensional structures of wildtype and mutant proteins were monitored by NMR spectroscopy.All five mutant HPrs had native conformations. The ATP-dependentHPr kinase can phosphorylate all HPr derivatives at Ser46. ThePTS activity of the amino-terminal HPr mutant proteins 17RHand 18PA was different compared to wild type HPr. In contrast,the car boxy-terminal mutant HPrs possessed a similar enzymeactivity to the wild type HPr. The 17RH and 18PA HPrs with substitutionnear the active centre His15 showed a very slow phosphorylationby enzyme I but the further transfer of the phosphoryl groupto enzyme III was also strongly inhibited. The enzyme activityof the HPr 17RH was significantly improved at low pH. NMR pH-titrationexperiments showed that Arg17 is not responsible for the lowpKa, of the active centre His15 but this positively chargedresidue is essential in this position for the HPr activity. 相似文献
16.
Saviranta P; Pajunen M; Jauria P; Karp M; Pettersson K; Mantsala P; Lovgren T 《Protein engineering, design & selection : PEDS》1998,11(2):143-152
We have employed random mutagenesis and phage display to improve the
steroid-specificity of an anti-17beta-estradiol Fab fragment. The VH domain
was mutated using error-prone PCR; the mutation rate was controlled by
adjusting the number of effective duplications. A phage library of 2 x
10(6) independent mutants was generated, each mutant containing on average
24 amino acid changes. We selected for decreased testosterone (TES)
cross-reactivity by adding a large excess TES as a competitor to the
panning reactions. After four panning rounds, the cross-reactivities of the
individual mutant clones ranged from 19 to 4%, showing up to 20-fold
improvement over the original value (78%). Estradiol affinities were mainly
unchanged. Sequencing of the VH regions revealed two hot spots, one located
around Ser32 in CDR1 and the other around Thr52A in CDR2, while no
mutations were found in CDR3. Although most clones had multiple mutations,
it was possible to deduce the residues relevant to the improved specificity
by comparing the sequences and binding data of the mutants. We demonstrated
that controlled error-prone PCR mutagenesis is a rapid method to identify
such key residues, lending itself to the scanning of 'lead' positions for
further mutagenesis by other methods.
相似文献
17.
In a previous directed evolution study, the B-FIT approach to increasing the thermal robustness of proteins was introduced and applied to the lipase from Bacillus subtilis. It is based on the general concept of iterative saturation mutagenesis (ISM), according to which sites in an enzyme are subjected to saturation mutagenesis, the best hit of a given library is then used as a template for randomization at other sites, and the process is continued until the desired catalyst improvement has been achieved. The appropriate choice of the ISM sites is crucial; in the B-FIT method the criterion is residues characterized by highest B factors available from X-ray crystallography data. In the present study, B-FIT was employed in order to increase the thermal robustness of the epoxide hydrolase from Aspergillus niger. Several rounds of ISM resulted in the best variant showing a 21 °C increase in the T(60)(50) value, an 80-fold improvement in half-life at 60 °C, and a 44 kcal mol(-1) improvement in inactivation energy. Seven other variants were also evolved with moderate yet significant improvements; these were characterized by 10-14 °C increases in T(60)(50), 20-30-fold improvement in half-lives at 60 °C and 15-20 kcal mol(-1) elevations in activation energy. Unexpectedly, in the ISM process the best variants were obtained from essentially neutral or even inferior mutant parents, that is, when a given library contains no improved mutants. This constitutes a practical way to escape from what appear to be local minima ("dead ends") in the fitness landscape-a finding of notable significance in directed evolution. 相似文献
18.
Mitra Kakoli; Steitz Thomas A.; Engelman Donald M. 《Protein engineering, design & selection : PEDS》2002,15(6):485-492
We have explored the interchangeability of soluble and membraneproteins by attempting to render a helical membrane protein`water soluble' through mutation of its lipid-exposed residues.Using an atomic resolution structure of bacteriorhodopsin (bR),two different strategies were developed to identify lipid-exposedresidues for mutation. In the first strategy all residues intrimeric bR with solvent accessibility >35% were marked forreplacement. Replacement residues were chosen so as to map anaverage surface of helical soluble proteins onto the bR surface,resulting in the mutagenesis of 14.9% of surface residues. Thesecond strategy took into account the observation that accessibleresidues can be categorized as fully or partially accessible.Consequently, three mutants were designed based on monomericbR, all with their accessible residues changed and with varyingextents of mutagenesis of partially accessible residues. 13.524.3%of the wild-type surface was altered in these designs. The constructfor the first design was cloned into Escherichia coli. Traceamounts of the mutant protein were expressed with the concurrentoverexpression of an endogenous prolyl isomerase. In contrast,all three mutant proteins of the second design expressed welland could be purified to homogeneity. Systematic refolding trialswere undertaken with limited success at solubilization in aqueousmedia. We have discussed the feasibility of applying the `solubilizationstrategy' outlined here to membrane proteins. 相似文献
19.
Introduction of polar and charged residues on the lipid-exposed face of
transmembrane proteins using site-directed mutagenesis represents a novel
approach to render membrane proteins more soluble in aqueous solution. We
have sequentially introduced as many as five polar and charged amino acids
onto the lipid-exposed face of helix D of bacteriorhodopsin from
Halobacterium salinarium. The most polar mutant (Q4D) has four glutamine
residues at positions 113, 116, 120 and 124 and an aspartate at position
117. In combination with wild-type residues Gln105, Thr107, Thr121 and
Thr128, the Q4D mutant has a nearly uninterrupted stripe of polar residues
on the surface of helix D. All of the mutants refold, bind retinal and the
resulting pigments exhibit light- and dark-adapted UV and visible
spectroscopic properties that are similar to the wild-type pigment,
indicating that the secondary, tertiary and active site structures are
similar to the wild-type protein. These results demonstrate that
micelle-solubilized bacteriorhodopsin can tolerate multiple
non-conservative substitution of amino acids that face the non-polar
portion of the lipid bilayer in vivo, thus lending credence to the notion
of partial or complete solubilization of integral membrane proteins by
site-directed mutagenesis.
相似文献
20.
Edge M; Forder C; Hennam J; Lee I; Tonge D; Hardern I; Fitton J; Eckersley K; East S; Shufflebotham A; Blakey D; Slater A 《Protein engineering, design & selection : PEDS》1998,11(12):1229-1234
Variants of human pancreatic carboxypeptidase B (HCPB), with specificity
for hydrolysis of C-terminal glutamic acid and aspartic acid, were prepared
by site-directed mutagenesis of the human gene and expressed in the
periplasm of Escherichia coli. By changing residues in the lining of the
S1' pocket of the enzyme, it was possible to reverse the substrate
specificity to give variants able to hydrolyse prior to C- terminal acidic
amino acid residues instead of the normal C-terminal basic residues. This
was achieved by mutating Asp253 at the base of the S1' specificity pocket,
which normally interacts with the basic side- chain of the substrate, to
either Lys or Arg. The resulting enzymes had the desired reversed polarity
and enzyme activity was improved significantly with further mutations at
residue 251. The [G251T,D253K]HCPB double mutant was 100 times more active
against hippuryl-L-glutamic acid (hipp-Glu) as substrate than was the
single mutant, [D253K]HCPB. Triple mutants, containing additional changes
at Ala248, had improved activity against hipp-Glu substrate when position
251 was Asn. These reversed-polarity mutants of a human enzyme have the
potential to be used in antibody-directed enzyme prodrug therapy of cancer.
相似文献