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.     

Downregulation of eRF1 by RNA interference increases mis-acylated tRNA suppression efficiency in human cells

The site-specific incorporation of non-natural amino acids into proteins by nonsense suppression has been widely used to investigate protein structure and function. Usually this technique exhibits low incorporation efficiencies of non-natural amino acids into proteins. We describe for the first time an approach for achieving an increased level of nonsense codon suppression with synthetic suppressor tRNAs in cultured human cells. We find that the intracellular concentration of the eukaryotic release factor 1 (eRF1) is a critical parameter influencing the efficiency of amino acid incorporation by nonsense suppression. Using RNA interference we were able to lower eRF1 gene expression significantly. We achieved a five times higher level of amino acid incorporation as compared with non-treated control cells, as demonstrated by enhanced green fluorescent protein (EGFP) fluorescence recovery after importing a mutated reporter mRNA together with an artificial amber suppressor tRNA.     

Red fluorescent protein variants with incorporated non-natural amino acid analogues

Fluorescent proteins are important tools in biotechnology applications and biosensing. DsRed, a red fluorescent protein, has expanded the colors of fluorescent proteins beyond the more commonly used green fluorescent protein. Many genetic modifications have been performed on DsRed to overcome some of its drawbacks. These primarily focused on overcoming the oligomerization detrimental to DsRed activity, and the parasitic green fluorescence caused by the immature chromophore. One such variant, DsRed-monomer, has minimal green fluorescence and no oligomerization. A few traditional mutagenesis studies have been done with DsRed and its mutants to shift the fluorescence wavelengths creating additions to the pallet of fluorescent protein colors. We have explored incorporation of non-natural amino acid analogues into DsRed-Monomer, obtaining variants with differing emission properties. In this work, two such analogues of tyrosine have been incorporated into DsRed-Monomer: 3-amino-l-tyrosine and 3-fluoro-l-tyrosine. Tyrosine analogues were chosen due to the role of tyrosine in the formation and structure of the protein's chromophore. The variants obtained in our study showed altered emission wavelengths and spectral characteristics. Our study demonstrates that incorporation of non-natural analogues into DsRed-Monomer is a viable approach to alter the spectral characteristics of the protein. We envision that this study will open up the door to non-natural mutagenesis studies with red fluorescent proteins and its mutants.     

Altering substrate preference of carboxypeptidase Y by a novel strategy of mutagenesis eliminating wild type background

To change the substrate preference of carboxypeptidase Y theputative substrate binding pocket was subjected to random mutagenesis.Based upon the three-dimensional structure of a homologous enzymefrom wheat, we hypothesized that Tyr147, Leu178, Glu215, Arg216,Ile340 and Cys341 are the amino acid residues of carboxypeptidaseY that constitute S₁ the binding pocket for the penultimateamino acid side chain of the substrate. We developed a new andgenerally applicable mutagenesis strategy to facilitate efficientscreening of a large number of mutants with multiple changesin carboxypeptidase Y. The key feature is the elimination ofwild type background by introducing a nonsense codon at eachtarget site for subsequent mutagenesis by degenerate oligonucleotides.The entire hypothesized S₁ binding pocket and subsets of itwere subjected to saturation mutagenesis by this strategy, andscreening yielded a number of mutant enzymes which have up to150 times more activity (k_cat/K_m towards CBZ-LysLeu-OH thanthe wild type enzyme. All selected mutants with increased activityhave mutations at position 178. Mutagenesis of positions 215and 216 has virtually no effect on the activity, while mutatingpositions 340 and 341 generally reduces activity.     

Directed evolution of subtilisin E in Bacillus subtilis to enhance total activity in aqueous dimethylformamide

Sequential rounds of error-prone PCR to introduce random mutationsand screenrng of the resultant mutant libraries have been usedto enhance the total catalytic activity of subtilisin E significantlyin a non-natural environment, aqueous dimethylformamide (DMF).Seven DNA substitutions coding for three new amino acid substitutionswere identified in a mutant isolated after two additional generationsof directed evolution carried out on 10M subtilisin E, previously‘evolved’ to increase its specific activity in DMF.A Bacillus subtilis-Escherichia coli shuttle vector was developedin order to increase the size of the mutant library that couldbe established in B.subtilis and the stringency of the screeningprocess was increased to reflect total as well as specific activity.This directed evolution approach has been extremely effectivefor improving enzyme activity in a non-natural environment:the resulting-evolved 13M subtilisin exhibits specific catalyticefficiency towards the hydrolysis of a peptide substrate succlnyl-Ala-Ala-Pro-Phe-p-nitroanilidein 60% DMF solution that is three times that of the parent 10Mand 471 times that of wild type subtilisin E. The total activityof the 13M culture supernatant is enhanced 16-fold over thatof the parent 10M.     

Alteration of product specificity of Aeropyrum pernix farnesylgeranyl diphosphate synthase (Fgs) by directed evolution

Directed evolution of the C25 farnesylgeranyl diphosphate synthase of Aeropyrum pernix (Fgs) was carried out by error-prone PCR with an in vivo color complementation screen utilizing carotenoid biosynthetic pathway enzymes. Screening yielded 12 evolved clones with C20 geranylgeranyl diphosphate synthase activity which were isolated and characterized in order to understand better the chain elongation mechanism of this enzyme. Analysis of these mutants revealed three different mechanisms of product chain length specificity. Two mutants (A64T and A64V) have a single mutation at the 8th amino acid upstream of a conserved first aspartate-rich motif (FARM), which is involved in the mechanism for chain elongation reaction of all prenyl diphosphate synthases. One mutant (A135T) carries a single mutation at the 7th amino acid upstream of another conserved region (141GQ142), which was recently found to be another important region controlling chain elongation of a type III C20 geranylgeranyl diphosphate synthase and Escherichia coli C15 farnesyl diphosphate synthase. Finally, one mutant carrying four mutations (V84I, H88R, I177 M and M191V) is of interest. Molecular modeling, site-directed mutagenesis and in vitro assays of this mutant suggest that product chain-length distribution can be also controlled by a structural change provoked by a cooperative interaction of amino acids.     

Structure and dynamics of staphylococcal nuclease mutants as studied by fluorescence quenching techniques

Fluorescence techniques have been used to investigate the effectof mutations on the structure and dynamics of staphylococcalnuclease. An estimate of the accessibility to acrylamide ofthe enzyme's single tryptophan residue (Trp140) was obtainedfrom the Stern–Volmer constant for fluorescence quenching.This was Indicative of a partially buried tryptophan in thewild-type nuclease. Five single-site mutant nucleases (H124L,V66L, G88V, G79S and F76V) and one double mutant (V66L + G88V),with widely differing stabilities to denaturants, gave Stern–Volmerconstants which were very similar to that of their parent enzyme.Studies of the temperature- and viscosity-dependence of quenchingsuggest that access by acrylamide to Trp140 is limited by diffusionrather than by protein structural fluctuations. Lifetime-resolvedfluorescence anisotropy studies using steady-state instrumentationsuggest that there is very little segmental motion of the Trp140;most of the anisotropy therefore decays due to protein rotationin the solution. Rotational correlation times for several nucleasemutants have been determined and these are very similar to thatof the native nuclease. Thus it appears that these substitutionsin the primary amino acid sequence, which have significant effectson the stability of the folded proteins, do not cause a significantchange in the protein structure or dynamics around Trp140.     

Site-directed mutagenesis and X-ray crystallography of two phospholipase A2 mutants: Y52F and Y73F

Tyr52 and Tyr73 are conserved amino acid residues throughoutall vertebrate phospholipases A₂. They are part of an extendedhydrogen bonding system that links the N-terminal -NH⁺₃ -groupto the catalytic residues His48 and Asp99. These tyrosines werereplaced by phenylalanines in a porcine pancreatic phospholipaseA₂ mutant, in which residues 62–66 had been deleted (62–66PLA₂).The mutations did not affect the catalytic properties of theenzyme, nor the folding kinetics. The stability against denaturatlonby guanidine hydrochloride was decreased, however. To analysehow the enzyme compensates for the loss of the tyrosine hydroxylgroup, the X-ray structures of the Y52F and AY73F mutants weredetermined. After crystallographic refinement the final crystallographicR-factors were 18.1% for the %Y52F mutant (data between 7 and2.3 Å resolution) and 19.1% for the Y73F mutant (databetween 7 and 2.4 Å resolution). No conformational changesoccurred in the mutants compared with the 62–66PLA₂, butan empty cavity formed at the site of the hydroxyl group ofthe former tyrosine. In both mutants the Asp99 side chain losesone of its hydrogen bonds and this might explain the observeddestabilization.     

Protein engineering of the high-alkaline serine protease PB92 from Bacillus alcalophilus: functional and structural consequences of mutation at the S4 substrate binding pocket

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 126–128.Substitution of Val102 by Trp results in a 12–fold 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 (‘NSA’mutant). 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 ‘NSA’mutant 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.     

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.     

Characterization of active-site aromatic residues in xylanase A from Streptomyces lividans

The role of four aromatic residues (W85, Y172, W266 and W274)in the structure–function relationship in xylanase A fromStreptomyces lividans (XlnA) was investigated by site-directedmutagenesis where each residue was subjected to three substitutions(W85A/H/F; W266A/H/F; W274A/H/F and Y172A/F/S). These four aminoacids are highly conserved among family 10 xylanases and structuraldata have implicated them in substrate binding at the activesite. Far-UV circular dichroism spectroscopy was used to showthat the overall structure of XlnA was not affected by any ofthese mutations. High-performance liquid chromatographic analysisof the hydrolysis products of birchwood xylan and xylopentaoseshowed that mutation of these aromatic residues did not alterthe enzyme's mode of action. As expected, though, it did reducethe affinity of XlnA for birchwood xylan. A comparison of thekinetic parameters of different mutants at the same positiondemonstrated the importance of the aromatic nature of W85, Y172and W274 in substrate binding. Replacement of these residuesby a phenylalanine resulted in mutant proteins with a K_M closerto that of the wild-type protein in comparison with the othermutations analyzed. The kinetic analysis of the mutant proteinsat position W266 indicated that this amino acid is importantfor both substrate binding and efficient catalysis by XlnA.These studies also demonstrated the crucial role of these activesite aromatic residues for the thermal stability of XlnA.     

Effect of Lys->Arg mutation on the thermal stability of Cu,Zn superoxide dismutase: influence on the monomer-dimer equilibrium

The thermal stability of two single (K3R, K67R) and one double(K3R-K67R) mutants of Xenopus laevis B Cu,Zn superoxide dismutasehas been studied to test LysArg substitution as an ‘electrostaticallyconservative’ strategy to increase protein stability.The K3R mutant displays an increased thermostability with respectto the wild-type enzyme, whilst a decreased stability was observedin the case of the K67R and K3R-K67R mutants. Concentrationdependence of the apparent inactivation constant (k_app) of thelatter mutants, as compared to that of the wild type enzymeand K3R mutant, indicates that their higher sensitivity to heatinactivation is due to a perturbation of the dimer association.These results are confirmed also by fluorescence anisotropymeasurements of the internal probe Tyr149. The possible roleof Arg67 in perturbing the dimer dissociation equilibrium towardthe monomeric form is discussed.     

On the choice of reference mutant states in the application of the double-mutant cycle method

Pairwise interactions in proteins can be detected and, in certaincircumstances, their strength measured by applying the methodof double-mutant cycles. Such cycles comprise wild type protein,two single mutants and the corresponding double mutant The analysisof double-mutant cycles is most straightforward when the mutationsare to alanine since interactions are mostly removed withoutnew interactions being formed. Here, ‘not-to-alanine’double-mutant cycles are analysed. It is shown that a ‘not-to-alanine’double-mutant cycle can be decomposed into four double-mutantcycles with mutations only to alanine. The coupling energy correspondingto the ‘not-to-alanine’ double-mutant cycle is expressedas a function of the coupling energies of these four cycles.     

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.     

A new method for random mutagenesis of complete genes: enzymatic generation of mutant libraries in vitro

A new efficient in vitro mutagenesis method for the generationof complete random mutant libraries, containing all possiblesingle base substitution mutations in a cloned gene is described.The method is based on controlled use of polymerases. Four populationsof DNA molecules are first generated by primer elongation sothat they terminate randomly, but always just before a knowntype of base (before A, C, G or T respectively). Each of thefour populations is then mutagenized in a separate misincorporationreaction, where the correct base can now be omitted. The regenerationof wild-type sequences can thus be efficiently avoided. Also,the misincorporating nucleotide concentrations can be optimizedto give the three possible single mutations in close to equalratio. The mutagenesis can be precisely localized within a predeterminedtarget region of any size, and vector sequences remain intact.We have mutagenized the DNA coding for the -fragment of Escherichiacoli ß-galactosidase, and identified 176 differentbase substitution mutations by sequencing. The present methodgives mutant yields of 40–60%, when the mutants containabout one amino acid change per protein molecule. All typesof base substitution mutations can be generated and deletionsare rare. The efficiency of this method permits the use of relativelyelaborate screening systems to isolate mutants of either structuralgenes or regulatory regions.     

The role of tyrosine residues in the DNA-binding site of the Pf1 gene 5 protein

The 144 amino acid gene 5 protein of bacteriophage Pf1 bindstightly and cooperatively to single-stranded DNA during replicationof the phage genome. It has been suggested that aromatic aminoacid side chains are important for this interaction, probablythrough base stacking with the DNA. We have analysed the accessibilityof tyrosine residues in the DNA—protein complex, and theirimportance to the DNA-binding activity of the protein, by chemicalmodification and protection experiments using tetranitromethane.Tyrosines 21, 30 and 55 are surface accessible in the free proteinbut are protected from modification in the complex with phageDNA. Moreover, modification of these residues in the free proteinabolishes the ability to bind to DNA or oligonucleotides, asjudged by fluorescence spectroscopy and gel retardation analysis.Modification of the protein also results in the formation ofan intersubunit covalent cross-link between Tyr55 and Phe76,suggesting that Phe76 is located within the DNA-binding cleftof the protein. It is proposed that residues 17–34 ofthe Pf1 gene 5 protein form a beta-hairpin analogous to the‘DNA-binding wing’ of the fd and Ike gene 5 proteins.We suggest the existence of a single-stranded DNA binding motif,in which Tyr30 of the Pf1 protein is equivalent to the functionallyimportant Tyr26 of the fd gene 5 protein.     

Directed molecular evolution by somatic hypermutation

After rearrangement of immunoglobulin gene segments, the immune system evolves the antibody repertoire by mutating the immunoglobulin variable region at a high rate. While this somatic hypermutation was thought to occur only at the variable region, recent studies suggest that hypermutation can occur at locations throughout the genome. Building upon this notion, we sought to exploit this mechanism as a mutagenesis tool. We created a substrate based on GFP that could be screened using flow cytometry and showed that retroviral infection can deliver the transgene to genomic locations that support hypermutation. Infected cells generated various GFP mutants with increased fluorescence intensity and analysis revealed mutations not only at the chromophore, but also an unexpected mutation at a distant residue. Our results demonstrate in principle that immunoglobulin somatic hypermutation can be a potent means of mutagenesis. With appropriate selection conditions it may be utilized to evolve gene products with desired properties.     

Hydrophobicity engineering of cholera toxin A1 subunit in the strong adjuvant fusion protein CTA1-DD

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.     

Concurrent mutations in six amino acids in beta-glucuronidase improve its thermostability

To achieve a thermostable beta-glucuronidase (GUS) and identify key mutation sites, we applied in vitro directed evolution strategy through DNA shuffling and obtained a highly thermostable mutant GUS gene, gus-tr, after four rounds of DNA shuffling and screening. This variant had mutations in 15 nucleic acid sites, resulting in changes in 12 amino acids (AAs). Using gus-tr as the template, we further performed site-directed mutagenesis to reverse the individual mutation to the wild-type protein. We found that six sites (Q493R, T509A, M532T, N550S, G559S and N566S) present in GUS-TR3337, were the key AAs needed to confer its high thermostability. Of these, Q493R and T509A were not reported previously as important residues for thermostability of GUS. Furthermore, all of these six mutations must be present concurrently to confer the high thermostability. We expressed the gus-tr3337 gene and purified the GUS-TR3337 protein that contained the six AA mutations. Compared with the wild-type protein which lost its activity completely after 10 min at 70 degrees C, the mutant GUS-TR3337 protein retained 75% of its activity when heated at 80 degrees C for 10 min. The GUS-TR3337 exhibited high activity even heated at 100 degrees C for 30 min on nitrocellulose filter. The comparison of molecular models of the mutated and wild-type enzyme revealed the relation of protein function and these structural modifications.     

Alteration of catalytic properties of chymosin by site-directed mutagenesis

Artificial mutations of chymosin by recombinant DNA techniqueswere generated to analyze the structure–function relationshipin this characteristic aspartk proteinase. In order to preparethe mutant enzymes in their active form, we established proceduresfor purification of correctly refolded prochymosin from inclusionbodies produced in Escherichia coli transformants and for itssubsequent activation. Mutagenesis by linker insertion intocDNA produced several mutants with an altered ratio of milkclotting activity to proteolytic activity and a different extentof stability. In addition to these mutants, several mutantswith a single amino acid exchange were also constructed by site-directedmutagenesis and kinetic parameters of these mutant enzymes weredetermined by using synthetic hexa- and octa-peptides as substrates.Exchange of Tyr75 on the flap of the enzyme to Phe caused amarked change of substrate specificity due to the change ofk_cat or K_m, depending on the substrate used. Exchange of Val110and Phe111 also caused a change of kinetic parameters, whichindicates functional involvement of these hydrophobic residuesin both the catalytic function and substrate binding. The mutantLys220–Leu showed a marked shift of the optimum pH tothe acidic side for hydrolysis of acid-denatured haemoglobinalong with a distinct increase in k_cat for the octa-peptidein a wide pH range.