Synthesis and sequence optimization of GFP mutants containing aromatic non-natural amino acids at the Tyr66 position

In order to alter the fluorescence properties of green fluorescent protein (GFP), aromatic non-natural amino acids were introduced into the Tyr66 position of GFP in a cell-free translation system using a four-base codon method. Two non-natural mutants (O-methyltyrosine and p-aminophenylalanine mutants) out of 18 mutants showed blue-shifted but weak fluorescence compared with wild-type GFP. Then the aminophenylalanine mutant was sequence optimized by introducing random mutations around the Tyr66 site. For this purpose, a method for random mutation of non-natural proteins in a cell-free system was developed. Three aminophenylalanine mutants with Y145F, Y145L and Y145 M mutations were obtained, which exhibited increased fluorescence by 1.5-, 3- and 4-fold, respectively. These results indicate that random mutation around non-natural amino acids is useful strategy in order to improve protein functions that are reduced by non-natural amino acid incorporation. The method described here will be applicable to other non-natural mutant proteins in a high-throughput manner.     

The Escherichia coli aspartate receptor: sequence specificity of a transmembrane helix studied by hydrophobic-biased random mutagenesis

The Escherichia coli aspartate receptor is a dimer with twotransmembrane sequences per monomer that connect a periplasmicligand binding domain to a cytoplasmic signaling domain. Themethod of 'hydrophobic-biased' random mutagenesis, that we describehere, was used to construct mutant aspartate receptors in whicheither the entire transmembrane sequence or seven residues nearthe center of the transmembrane sequence were replaced withhydrophobic and polar random residues. Some of these receptorsresponded to aspartate in an in vivo chemotaxis assay, whileothers did not. The acceptable substitutions included hydrophobicto polar residues, small to larger residues, and large to smallerresidues. However, one mutant receptor that had only a few hydrophobicsubstitutions did not respond to aspartate. These results addto our understanding of sequence specificity in the transmembraneregions of proteins with more than one transmembrane sequence.This work also demonstrates a method of constructing familiesof mutant proteins containing random residues with chosen characteristics.     

Improvement in the alkaline stability of subtilisin using an efficient random mutagenesis and screening procedure

An efficient random mutagenesis procedure coupled to a replicaplate screen facilitated the isolation of mutant subtilisinsfrom Bacillus amyloliquefaciens that had altered autolytic stabilityunder alkaline conditions. Out of about 4000 clones screened,approximately 70 produced subtilisins with reduced stability(negatives). Two dones produced a more stable subtilisin (positives)and were identified as having a single mutation, either IIe107Valor Lys2l3Arg (the wild-type amino acid is followed by the codonposition and the mutant amino acid). One of the negative mutants,Met50Val, was at a site where other homologous subtilisins containeda Phe. When the Met50Phe mutation was introduced into the B.amyloliquefaciens gene, the mutant subtilisin was more alkalinestable. The double mutant IIe107Val/Lys2l3Arg) was more stablethan the isolated single mutant parents. The triple mutant (Met50Phe/IIel07Val/Lys2l3Arg)was even more stable than IIe107Val/Lys2l3Arg (up to two timesthe autolytic half-time of wild-type at pH 12). These studiesdemonstrate the feasibility for improving the alkaline stabilityof proteins by random mutagenesis and identifying potentialsites where substitutions from homologous proteins can improvealkaline stability.     

Position-dependent protein mutant profile based on mean force field calculation

The application of the mean force field in protein mutant stabilityprediction is explored. Based on protein main chain characteristics,including polar fraction, accessibility and dihedral angles,the mean force field was constructed to evaluate the compatibilitybetween an amino acid residue and its environment, from whicha position-dependent protein mutant profile was constructed.At each position along a protein sequence, the native residuewas replaced by the other 19 types of amino acid residues. Thematches were evaluated by energies from mean force field calculation,from which a mutant profile along the protein sequence was derived.General characteristics of such a profile were analyzed. Mutantstabilities for two sets of mutants in two proteins were foundto be reasonable compared with experimental data, which indicatesthat the present method can act as a guide in protein engineeringand as an effective scoring matrix in protein sequence–structurealignment studies.     

Site-directed mutagenesis to fine-tune enzyme specificity

We have used a combination of a genetic selection and oligonucleotide-directedmutagenesis to introduce a series of amino add replacementsfor a single residue into Escherichia coliglutaminyl-tRNA synthetase.The mutant enzymes mischarge supFtRNA^Tyr, with glutamine, tovarying degrees depending on the polarity of the side chainintroduced but apparently not depending on the size or shapeof the side chain. These results indicate that repulsive charge-chargeinteractions may be important for specific recognition of nucleicacids by proteins and illustrate how a mutant, derived fromgenetic selection, may be further modified in activity by oligonucleotide-directedmutagenesis.     

Involvement of various amino- and carboxyl-terminal residues in the active site of the histidine-containing protein HPr of the phosphoenolpyruvate-dependent phosphotransferase system of Staphylococcus carnosus: site-directed mutagenesis with the ptsH gene, biochemical characterization and NMR studies of the mutant proteins

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 lowpK_a, of the active centre His15 but this positively chargedresidue is essential in this position for the HPr activity.     

A simple dual selection for functionally active mutants of Plasmodium falciparum dihydrofolate reductase with improved solubility

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.     

Cassette mutagenesis of Met121 in azurin from Pseudomonas aeruginosa

Cassette mutagenesis was used to exchange the suggested copperligand Met121 in azurin to all other amino acids, and a stopcodon. The mutant proteins were characterized by optical absorptionspectroscopy and EPR. At low pH, all mutants exhibit the characteristicsof a blue type 1 copper protein, indicating that methionineis not needed to create a blue copper site. At high pH, theGlu121 and the Lys121 mutants constitute a new form of protein-boundcopper that is outside the range of type1 copper.     

Double-site ricin B chain mutants retain galactose binding

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 0.01–0.2 mg/1. The purityafter monoclonal antibody affinity chromatography was 1–20%.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(25–75%) 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.     

Selection of single domain binding proteins by covalent DNA display

Selection technologies such as phage and ribosome display, which provide a physical linkage between genetic information and encoded polypeptide, are important tools for the engineering of proteins for diagnostic and therapeutic applications. We have recently described a selection strategy called covalent DNA display, in which individual proteins are covalently linked to the cognate encoding DNA template in separate droplets of a water-in-oil emulsion. We here report on the optimization of several experimental steps in covalent DNA display technology, such as the elution conditions and the PCR strategy used for the amplification of selected DNA templates. A PCR assembly strategy was developed, which allows the amplification of the DNA templates over repeated rounds of selection. In addition, we could demonstrate that approximately 50% of the DNA templates form a covalent adduct with the corresponding proteins in the compartments of a water-in-oil emulsion. In model selection experiments, differences in recovery efficiency <100 000 per round of selection could be observed when comparing a specific binding polypeptide with a binder of irrelevant specificity. Furthermore, the optimized protocol was successfully applied for the selection of single domain proteins, capable of specific binding to mouse serum albumin (MSA). A mutant derived from the SH3 domain of the Fyn kinase, with millimolar affinity to MSA, was affinity matured using covalent DNA display and yielded several MSA binding FynSH3 variants with dissociation constants in the 100 nM range.     

Insertional re-activation of a chloramphenicol acetyltransferase misfolding mutant protein

The deletion of nine residues from the C-terminus of the bacterialchloramphenicol acetyltransferase (CAT) results in depositionof the mutant protein in cytoplasmic inclusion bodies and lossof chloramphenicol resistance in Escherichia coli. This foldingdefect is relieved by C-terminal fusion of the polypeptide withas few as two residues. Based on these observations, efficientpositive selection for the cloning of DNA fragments has beendemonstrated. The cloning vector encodes a C-terminally truncatedCAT protein. Restriction sites in front of the stop codon allowthe insertion of target DNA, resulting in the production ofproperly folded CAT fusion proteins and regained chloramphenicolresistance. The positive selection of recombinants is accomplishedby growth of transformants on chloramphenicol-containing agarplates. The method appears particularly convenient for the cloningof DNA fragments amplified by the PCR because minimal informationto restore CAT folding can be included in the primers. The cloningof random sequences shows that the folding defect can be relievedby fusion to a wide variety of peptides, providing great flexibilityto the positive selection system. This vector may also contributeto the determination of the role of the C-terminus in CAT folding.     

A new substitution matrix for protein sequence searches based on contact frequencies in protein structures

The instabilities of the native structures of mutant proteinswith an amino acid exchange are estimated by using the contactenergy and the number of contacts for each type of amino acidpair, which were estimated from 18 192 residue–residuecontacts observed in 42 crystals of globular proteins. Theywere then used to evaluate a transition probability matrix ofcodon substitutions and a log relatedness odds matrix, whichis used as a scoring matrix to measure the similarity betweenprotein sequences. To consider amino acid substitutions in homologousproteins, base mutation rates and the effects of the geneticcode are also taken into account. The average fitness of anamino acid exchange is approximated to be proportional to thestructural stability of the mutant protein, which is then approximatedby the average energy change of the protein native structureexpected for the ammo acid exchange with neglect of the energychange of the denatured state. In global and local homologysearches, this scoring matrix tends to yield significantly higheralignment scores than either the unitary matrix or the geneticcode matrix, and also may yield higher alignment scores fordistantly related protein pairs than MDM78. One of advantagesof this scoring matrix is that the equilibrium frequencies ofcodons and also base mutation rates can be adjusted.     

Thermostable variants of bovine {beta}-lactoglobulin

The thermal stability of bovine ß-lactoglobulin (BLG)has been enhanced by the introduction of an additional disulfidebond. Wild-type BLG has two disulfide bonds, C106–C119and C66–C160, with a free cysteine at position 121. Wehave designed, with the aid of molecular modeling calculations,two mutants of a recombinant BLG (rBLG), L104C and A132C. Moleculardynamics simulations were performed at 300K to study the effectof these alterations on the conformation of the protein. Thesemutants were then created by site-directed mutagenesis and purifiedfrom Escherichia coli carrying a tac expression vector usinga two-step renaturation method. Formation of disulfide linkagesin the correct arrangement, as designed, was confirmed by peptidemapping. In contrast to wild-type rBLG, which polymerizes attemperatures >65°C, neither of the mutant proteins polymerized.The conformational stability of the L104C and A132C mutant proteinsagainst thermal denaturation has been substantially increased(8- 10°C) as compared with wild-type rBLG. Furthermore,the A132C rBLG exhibits an enhanced stability against denaturationby guanidine hydrocnloride as compared with the wild-type orL104C rBLG     

Solubilization of aggregation-prone heterologous proteins by covalent fusion of stress-responsive Escherichia coli protein, SlyD

The proteome profile of Escherichia coli BL21(DE3) generated in response to heat shock stress was analyzed by two-dimensional electrophoresis (2-DE), wherein we identified a FKBP-type peptidyl-prolyl cis-trans isomerse (PPIases), SlyD, as a stress-responsive (i.e. aggregation-resistant) protein. Even under an imposed severe stress condition where 29 out of 858 soluble proteins were totally eliminated and the synthesis levels of 171 proteins decreased over 5-fold, a 3.37-fold increase induced by heat shock treatment was observed in the synthesis level of SlyD compared with a non-stress condition. As a fusion partner, as well as solubility enhancer, SlyD facilitated folding and significantly increased the solubility of many aggregation-prone heterologous proteins in E. coli cytoplasm. SlyD was very effective in sequestering interactive surfaces of heterologous proteins associated with non-specific protein-protein interactions and the formation of inclusion bodies, most likely as a result of intrinsic folding efficiencies and/or chaperone-like activities. SlyD was also shown to be suitable for the production of a biologically active fusion mutant of Pseudomonas putida cutinase that is of considerable biotechnological and commercial interest.     

Toxicity-based selection of Escherichia coli mutants for functional recombinant protein production: application to an antibody fragment

We propose a novel approach to the selection of Escherichia coli bacterial strains improved for the production of recombinant functional proteins. This approach is based on aggregation-induced toxicity of recombinant proteins. We show that selection of clones displaying a reduced toxicity is an efficient means of isolating bacteria producing recombinant protein with reduced aggregation in favour of correct folding. For an efficient selection, we found that time of toxicity induction must be precisely determined and recombinant protein must be expressed as a fusion with a protein whose activity is easily detectable on plates, thus allowing elimination of non-productive mutants. Choosing the expression to the periplasmic space of an scFv fragment fused to the N-terminus of alkaline phosphatase as a model, we selected chromosomal mutations that reduce aggregation-induced toxicity and showed that they concomitantly improve production of a functional recombinant hybrid. The effects of the mutations isolated could then be cumulated with those of other strategies used for recombinant scFv production. Thus, we could ensure a 6- to 16-fold increase in production of a functional scFv-PhoA hybrid. This is the first report demonstrating the possibility of directly selecting on agar plates E.coli strains improved for functional recombinant protein production from a large bacterial mutant library.     

Deletions of single extracellular loops affect pH sensitivity, but not voltage dependence, of the Escherichia coli porin OmpF

The molecular basis for the voltage and pH dependence of the Escherichia coli OmpF porin activity remains unknown. The L3 loop was previously shown not be involved in voltage dependence. Here we used seven OmpF mutants where single extracellular loops, except L3, were deleted one at a time. The proteins are expressed at levels comparable to wild-type and purified as trimers. Wild-type and mutant proteins were inserted into planar lipid bilayers for electrophysiological measurement of their activity. Current-voltage relationships show the typical porin channel closure at voltages greater than the critical voltage. Measurements of critical voltages for the seven deletion mutants showed no significant differences relative to wild-type, hence eliminating the role of single loops in voltage sensitivity. However, deletions of loops L1, L7 or L8 affected the tendency of channels to close at acidic pH. Wild-type channels close more readily at acidic pH and their open probability is decreased by approximately 60% at pH 4.0 relative to pH 7.0. For mutants lacking loop L1, L7 or L8, the channel open probability was found not to be significantly different at pH 4.0 than at pH 7.0. The other deletion mutants retained a pH sensitivity similar to the wild-type channel. Possible mechanistic scenarios for the voltage- and pH dependence of E.coli OmpF porin are discussed based on these results.     

Effect of Glu-143 and His-231 substitutions on the catalytic activity and secretion of Bacillus subtilis neutral protease

On the basis of the homology with the Bacillus thermoproteolyticuszinc endopeptidase thermotysin, we hypothesized that Glu-143and His-231 are the key residues for the catalytic activityof the Bacillus subtilis neutral protease. To test this possibilityby site-directed mutagenesis, we substituted these two residueswith Ala, Ser, Trp and Arg, and Leu, Val and Cys respectively.All these substitutions dramatically affected the amount ofsecreted mutant proteins, as determined by immunological methods,and their catalytic activities. No appreciable secretion wasobserved with the three Glu mutants Trp, Ser and Arg, whereasthe Glu–Ala mutant enzyme was secreted at a level of afew hundred micrograms per litre of culture. The His mutantswere all secreted at higher levels (in the order of a few milligramsper litre) and their residual catalytic activity could be determinedusing Z-Ala-Leu-Ala as substrate. Our results confirm the keyrole played by Glu-143 and His-231 in catalysis and moreoversuggest the existence of a relationship between the catalyticactivity of the enzyme and the extent of its secretion. In thiscontext, we present data suggesting an autoproteolytic mechanismof cleavage of the precursor form of the enzyme, analogous tothe one previously reported for the B.subtilis subtilisin.     

Generation of a Ni(II) binding site by introduction of a histidine cluster in the structure of human glutathione transferase Al-1

Two mutant forms of human glutathione transferase (GST) Al–1with affinity for metal ions were constructed by introductionof His residues by site–directed mutagenesis. A mutant,2–His, contained the mutations Lys84Gln, Asp85His andGlu88His, and another, 5–His, contained the mutationsTyr79His, Asn80His, Lys84His, Asp85His and Glu88HLs. The mutantproteins were obtained in good yields (40–150 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 (2–Hismutant) did not bind as tightly to immobilized Nifll) as didthe mutant with five novel His residues (5–His mutant).When tested for affinity to immobilized Zn(II), only the 5–Hismutant remained bound to the column. The affinity of the 5–Hismutant for Ni(II) ions in solution was determined by bindingexperiments in an aqueous polymeric two–phase system.Analysis of the binding curve showed two binding sites per enzymesubunit and a dissociation constant of 6.7 1 . 6 M. The kineticconstants k_cat, K_m and k_cat/k_m for the reaction with glutathioneand l–chloro–2,4–dinitrobenzene were determinedby steady–state kinetic analysis and the parameter valuesfor the mutant forms were found to be indistinguishable fromthose obtained for the wild–type GST Al–1. The differencesin surface charge in the mutant proteins as compared with thewild–type enzyme did not alter the pH dependence of k_cat.The results provide an alternative method for purification offully active recombinant GST Al–1 by the introductionof novel metal binding sites. The data also showed that twoHis residues are sufficient for Ni(II) binding.     

Predicting local structural changes that result from point mutations

Point mutations are frequently used to explore the structureand/or function of proteins. The ability to predict the structuraleffects of point mutations would make the planning of such experimentsmore reliable. We have now derived a set of detailed predictiverules based on the comparison of crystal structures of pointmutants and wild types in 83 cases. Despite the surprising simplicityof these rules, they describe well the conformational changesin 85% of all point mutant structures available at present.     

Identification of interaction site of pseudoazurin with its redox partner, copper-containing nitrite reductase from Alcaligenes faecalis S-6

Pseudoazurin, a low molecular weight protein containing a singletype I copper, functions as an electron donor to a copper-containingnitrite reductase (NIR) in a denitrifying bacterium Alcaligenesfaecalis S-6. To elucidate the proteinprotein interaction betweenthese two copper-containing proteins, each of nine out of 13lysine residues on the surface of pseudoazurin were independentlyreplaced by alanine or aspartate, and the effects of the mutationson the interaction with NIR, as well as the physicochemicalproperties of pseudoazurin, were analyzed. All of the mutatedpseudoazurins showed optical spectra and oxidation-reductionpotentials almost identical to those of wildtype pseudoazurin,suggesting that none of the replacements of these lysine residuesaffected the environment around the type I copper site. Kineticanalysis of electron transfer between mutated pseudoazurinsand NIR reveals that the lysine mutations have very little effecton the rate of electron transfer to NIR, but substitution atresidues 10, 38, 57 and 77, all close to the copper site, substantiallydecreases the affinity of pseudoazurin for NIR. This suggeststhat pseudoazurin interacts with NIR through the region closeto the type I copper site. The refined X-ray structures of Lys38Aspand Lys10Asp/Lys38Asp show that the molecular structure hasindeed changed little. A new space group is observed for theLys109Ala mutant crystal. Crystal packing interactions changefor the Lys10Asp/Lys38Asp mutant but remain the same for Lys38Aspand Lys59Ala mutants.