A novel random mutagenesis approach using human mutagenic DNA polymerases to generate enzyme variant libraries

The in vitro MutaGen procedure is a new random mutagenesis method based on the use of low-fidelity DNA polymerases. In the present study, this technique was applied on a 2 kb gene encoding amylosucrase, an attractive enzyme for the industrial synthesis of amylose-like polymers. Mutations were first introduced during a single replicating step performed by mutagenic polymerases pol beta and pol eta. Three large libraries (>10(5) independent clones) were generated (one with pol beta and two with pol eta). The sequence analysis of randomly chosen clones confirmed the potential of this strategy for the generation of diversity. Variants generated by pol beta were 4-7-fold less mutated than those created with pol eta, indicating that our approach enables mutation rate control following the DNA polymerase employed for mutagenesis. Moreover, pol beta and pol eta provide different and complementary mutation spectra, allowing a wider sequence space exploration than error-prone PCR protocols employing Taq polymerase. Interestingly, some of the variants generated by pol eta displayed unusual modifications, including combinations of base substitutions and codon deletions which are rarely generated using other methods. By taking advantage of the mutation bias of naturally highly error-prone DNA polymerases, MutaGen thus appears as a very useful tool for gene and protein randomisation.     

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.     

Synthesis and mutagenesis of an IgG-binding protein based upon protein A of Staphylococcus aureus

A novel protein able to bind with high affinity to the Fc fragmentof IgG from a variety of animals has been produced by a genesynthesis approach. The IgG binding is accomplished by the presenceof a single or two consecutive domains based upon domain B fromprotein A of Staphylo-coccus aureus. The IgG-binding moietyis fused to a peptide containing 21, 53 or 81 amino acids derivedfrom the N-terminus of bovine DNase I. The latter is presentto guide the expression of the protein in Escherichia coli intoan inclusion body. This facilitates the high expression andrecovery of the IgG-binding domains. The binding activity ofthis fusion protein is very close to that of the native proteinA. Site-directed mutagenesis of the fusion protein and subsequentidentification of changed binding interactions is reported.     

Improved peptide function from random mutagenesis over short 'windows'

We have applied random mutagenesis over short contiguous residuetracts (‘windows’) within an active peptide (the-peptide of ß-galactosidase) such that all windowresidues are replaced simultaneously. A novel technique usingmixed synthetic oligonucleotides and selection against an EcoKrestrictionsite has allowed the construction of libraries of mutants fortwo separate windows, sites A and B. Mutant phenotypes can beeasily assessed in vivoby a complementation test, and panelsof mutants have been quantitatively tested in vivoThis allowedthe rapid probing of structural requirements for each site.The two windows yielded markedly disparate results. Site B wasmuch less stringent in its sequence requirements for significantfunction than Site A, and mutants with improved function wereisolated at Site B alone. In addition, one Site B mutant withwild-type levels of activity showed enhanced stability to heator a protein denaturant. We propose that short tracts with thecharacteristics of Site B constitute ‘secondary’interaction sites which are more tolerant of sequence diversity.Random manipulation of such secondary sites is thus more likelyto yield up-mutations for standard or altered environments.Window mutagenesis can in principle be applied to any protein-proteinor protein-Ugand interaction.     

Removal of an inter-domain hydrogen bond through site-directed mutagenesis: role of serine 176 in the mechanism of papain

A mutant of papain, where an inter-domain hydrogen bond betweenthe side chain hydroxyl group of a serine residue at position176 and the side chain carbonyl oxygen of a glutamine residueat position 19 has been removed by site-directed mutagenesis,has been produced and characterized kinetically. The mutationof Ser176 to an alanine has only a small effect on the kineticparameters, the k_cat/K_m for hydrolysis of CBZ-Phe-Arg-MCA bythe Serl76Ala enzyme being of 8.1 x 10⁴ /M/s compared with 1.2x 10⁵ /M/s for papain. Serine 176 is therefore not essentialfor the catalytic functioning of papain, even though this residueis conserved in all cysteine proteases sequenced. The pH-activityprofiles were shown to be narrower in the mutant enzyme by upto 1 pH unit at high ionic strength. This result is interpretedto indicate that replacing Ser 176 by an alanine destabilizesthe thiolate—imidazolium form of the catalytic site Cys25-Hisl59residues of papain. Possible explanations for that effect aregiven and the role of a serine residue at position 176 in papainis discussed.     

Biased random mutagenesis of peptides: determination of mutation frequency by computer simulation

Cassette mutagenesis is a method of protein engineering whichgenerates a wide diversity of genetic variants that can be subjectedto either selection or screening. As long as the target sequenceto be modified is kept short (corresponding to four to six aminoacids), complete combinatorial libraries can be produced. Amajor problem arises when longer peptides are to be engineeredfor desired functions. In such situations the production ofa limited collection of variants can be helpful; thus, biasedrandom mutagenesis and ‘doping schemes’ have beenreported previously. Here we describe a computer algorithm thatenables the determination of the degree of phosphoramidite contaminationof nucleotide precursor reservoirs. Through simulation of biologicaltranslation, the algorithm allows the prediction of the effectof contamination levels on the number of mutations to occurfor any given peptide sequence. In this study the cholinergicbinding site was used as a model sequence (22 amino acids).Considerations, based on the computer program, are discussedregarding the efficient design of phage-display combinatoriallibraries.     

Site-directed mutagenesis of aspartic acid 372 at the ATP binding site of yeast phosphoglycerate kinase: over-expression and characterization of the mutant enzyme

A new phosphoglycerate kinase over-expression vector, pYE-PGK,has been constructed which greatly facilitates the insertionand removal of mutant enzyme genes by cleavage at newly introducedBamtHI sites. This vector has been used to prepare mutant proteinin appreciable (100 mg) quantities for use in kinetic, crystaUographicand NMR experiments. Aspartate 372 is an invariant amino acidresidue in genes known to code for a functionally active PGK.The function of this acidic residue appears to be to help desolvatethe magnesium ion compfexed with either ADP or ATP when thissubstrate binds to the enzyme. Both crystallographk and nuclearmagnetic resonance experiments show that the replacement ofthe residue with asparagine has only minimal effects on theoverall structure. The substitution of the charged carboxylgroup with that of the neutral amide affects the binding ofthe nucleotide substrate as predicted but not, as might havebeen expected, the binding of 3-phospho-glycerate. The overallvelocity of the enzymic reaction (V_max) is reduced 10-fold bythe substitution of aspartic acid 372 by an asparagine residue(D372N). This reduction in V_max is considerably less than onewould expect from its known position within the structure ofthe enzyme. This result therefore poses questions about ourunderstanding of charged groups at the active centres of enzymesand of the reason for their apparent conservation.     

Expression of synthetic genes coding for completely new, nutritionally rich, artificial proteins

Synthetic genes (A, AB and AHB) constructed and cloned intopKK233-2 vector were recloned from the parent plasmid into thenew procaryotic expression vectors pGFY221N and pBIO52. GeneAF^–B (coding for all amino acids besides phenylalanine)was obtained by ‘cassette mutagenesis’ from geneAB. The plasmid pGFY221N was constructed from pGFY218L by replacingthe PstI by an NcoI site; plasmid pBIO52 was derived from pGFY221Nthrough replacing the 221-bp EoRl/NcoI fragment with a syntheticDNA segment of 52 bp representing the Escherichia coli atpEgene translational initiation region. The genes A, AB, AHB andAF^–B in the vector pGFY221N were expressed with a six-amino-acid-longleader sequence; in pBIO52 the genes were expressed directly.in vitro expression experiments were successful with all thegenes except with the AHB gene integrated into pGFY221N. Inthe E. coli minicell system expression was demonstrated withthe A gene in pGFY221N and the AF^–B and AHB genes in pBIO52.Complete translation of the expressed genes AB, AF^–B andAHB in either the in vitro or in vivo systems could be shownby using ³⁵S-labelled N-terminal methionine and C-terminal cysteine.Both amino acids occur only once in the peptide sequences.     

A new method for predicting binding affinity in computer-aided drug design

A new semi–empirical method for calculating free energiesof binding from molecular dynamics (MD) simulations is presented.It is based on standard thermodynamic cycles and on a linearapproximation of polar and non–polar free energy contributionsfrom the corresponding MD averages. The method is tested ona set of endothiapepsin inhibitors and found to give accurateresults both for absolute as well as relative free energies.     

Frame shuffling: a novel method for in vitro protein evolution

We describe 'frame shuffling', a novel method for preparing artificial protein libraries. With this method, a Y-family DNA polymerase known to introduce frame shift mutations at high rates is utilized to scramble the reading frames of a parental gene. The resultant progeny produce mutant proteins having segmental sequence changes. Such frame-shuffled mutant proteins exhibit physicochemical properties that differ from those of proteins obtained using conventional mutagenesis.     

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.     

A general method for relieving substrate inhibition in lactate dehydrogenases

The mutation S163L in human heart lactate dehydrogenase removessubstrate inhibition while only modestly reducing the turnoverrate for pyruvate. Since this is the third enzyme to show thisbehaviour, we suggest that the S163L mutation is a general methodfor the removal of substrate inhibition in L-LDH enzymes. Engineeringsuch enzymatic properties has clear industrial applicationsin the use of these enzymes to produce enantiomerically pure-hydroxy acids. The mutation leads to two principal effects.(1) Substrate inhibition is caused by the formation of a covalentadduct between pyruvate and the oxidized form of the cofactor.The inability of S163L mutants to catalyse the formation ofthis inhibitory adduct is demonstrated. However, NMR experimentsshow that the orientation of the nicotinamide ring in the mutantNAD⁺ binary complex is not perturbed. (2) The mutation alsoleads to a large increase in the K_M for pyruvate. The kineticand binding properties of S163L LDH mutants are accounted forby a mechanism which invokes a non-productive, bound form ofthe cofactor. Molecular modelling suggests a structure for thisnon-productive enzyme–NADH complex.     

A system based on specific protein-RNA interactions for analysis of target protein-protein interactions in vitro: successful selection of membrane-bound Bak-Bcl-xL proteins in vitro

Ribosome display systems are very effective and powerful tools for in vitro screening of transcribed mRNAs that encode proteins (or peptides) with specific (known or unknown) functions. We have modified such a system by exploiting the interaction between a tandemly fused MS2 coat-protein (MSp) dimer and the RNA sequence of the corresponding specific binding motif, C-variant (or Cv). We placed the MSp dimer at the N-terminus of a nascent protein and the Cv binding motif was attached to the 5' end of the protein's mRNA. This configuration enhanced the stability of the ribosome-mRNA complex. We demonstrate here that this improved ribosome display system provides an effective method for identifying the gene for a protein that binds to a protein of interest. We visualized the formation of polysome complexes in this advanced polysome display by atomic force microscopy (AFM) and found that the AFM images of polysomes in our system were different from those observed in the case of conventional ribosome display systems. Our results suggest that our technology might usefully complement yeast two-hybrid assays.     

Site-directed mutagenesis of pseudoazurin from Alcatigenes faecalis S-6; Pro80Ala mutant exhibits marked increase in reduction potential

Pseudoazurin (a blue copper protein or cupredoxin) of a denitrifyingbacterium Alcaligenes faecalis S-6 is a direct electron carrierfor a Cu-containing nitrite reductase (NIR) of the same organism.Site-directed mutagenesis of the pseudoazurin was carried outusing an Escherichia coli expression system. Replacement ofTyr74 by Phe to remove an internal hydrogen bond in the ß-barrelcaused a slight decrease in heat stability as well as a requirementfor a higher concentration of Cu²⁺ for production in the E.colihost. Exchange of Ala for Pro80 adjacent to His81, one of thefour ligands binding a type I Cu atom, caused a marked increasein reduction potential by 139 mV without change in the opticalabsorption spectrum. The ability of the pseudoazurin to transferelectrons to NIR was markedly diminished but the apparent K_mof NIR for pseudoazurin was not affected by the mutation. X-raydiffraction data collected on the oxidized and reduced formsof the Pro80Ala mutant show that a water molecule occupies thepocket created by the absent side chain. This observation suggeststhat the increase in reduction potential may be caused due tothe increased solvent accessibility to the Cu atom. The electrondensity difference maps on these structures (at 2.0 Å)show that this water moves during the change in oxidation state,and that there are small, but localized, conformational changes>6.5 Å from the copper site, as well as movement ofboth the Cu²⁺ and the cysteinate sulfur.     

Local polarity analysis: a sensitive method that discriminates between native proteins and incorrectly folded models

The evaluation of calculated protein structures is an importantstep in the protein design cycle. Known criteria for this assessmentof proteins are the polar and apolar, accessible and buriedsurface area, electrostatic interactions and other interactionsbetween the protein atoms (e.g. HO, S-S),atomic packing, analysisof amino acid environment and surface charge distribution. Weshow that a powerful test of accuracy of protein structure canbe derived by analysing the water contact of atoms and additionallytaking into account their polarity. On the basis of estimatedreference values of the polar fraction of typical globular proteinswith known structure (mean, SD and distribution), the evaluationof misfolded structures can be improved significantly. The referencevalues are derived by moving windows of different length (3–99amino acid residues) over the amino acid sequence. Model proteins,which are included in the Brookhaven protein structure databank,deliberately misfolded proteins, hypothetical proteins and predictedprotein structures are diagnosed as at least partially incorrectlyfolded. The local fault, mostly observed, is that polar groupsare buried too frequently in the interior of the protein. Thedatabase-derived quantities are useful in screening the designedproteins prior to experimentation and may also be useful inthe assessment of errors in the experimentally determined proteinstructures.     

Context dependence of phenotype prediction and diversity in combinatorial mutagenesis

Two different combinatorial mutagenesis experiments on the light-harvestingII (LH2) protein of Rhodobacter capsulatus indicate that heuristicrules relating sequence directly to phenotype are dependenton which sets or groups of residues are mutated simultaneously.Previously reported combinatorial mutagenesis of this chromogenicprotein (based on both phylogenetic and structural models) showedthat substituting amino acids with large molar volumes at Gly^ß31caused the mutated protein to have a spectrum characteristicof light-harvesting I (LH1). The six residues that underwentcombinatorial mutagenesis were modeled to lie on one side ofa transmembrane -helix that binds bacteriochlorophyll. In asecond experiment described here, we have not used structuralmodels or phylogeny in choosing mutagenesis sites. Instead,a set of six contiguous residues was selected for combinatorialmutagenesis. In this latter experiment, the residue substitutedat Gly^ß31 was not a determining factor in whetherLH2 or LH1 spectra were obtained; therefore, we conclude thatthe heuristic rules for phenotype prediction are context dependent.While phenotype prediction is context dependent, the abilityto identify elements of primary structure causing phenotypediversity appears not to be. This strengthens the argument forperforming combinatorial mutagenesis with an arbitrary groupingof residues if structural models are unavailable.     

GRAFTER: a computational aid for the design of novel proteins

The GRAFTER suite of programs provides geometric search andevaluation functions that simplify and automate the processof identifying the best scaffolds for a particular structuralmotif. Three applications of the GRAFTER suite are presented.Potential grafts between repressor and 434 repressor were identifiedthat should change the DNA binding specificity of these repressors.These results are compared with site-directed mutagenesis experimentsthat have been shown to alter repressor-DNA binding specificity.Next, 26 loops from antibody structures were grouped into familiesof similar structure. Grafts of antibody loops onto a pre-existingscaffold are an essential component of antibody humanization.Finally, interleukin (lL)-4 was searched as a scaffold thatmight accept the graft of a five residue epitope from humangrowth hormone (hGH). The existence of a crystal structure ofthe hGH-hGH receptor complex, extensive mutagenesis studiesof the hGH residues that contribute to the energetics of ligand-receptorinteractions and the gross structural homology between hGH andIL-4 make this an appealing computational target. The approachpresented here could aid the development of novel enzymes andbinding proteins     

A novel method for predicting transmembrane segments in proteins based on a statistical analysis of the SwissProt database: the PRED-TMR algorithm

We present a novel method that predicts transmembrane domainsin proteins using solely information contained in the sequenceitself. The PRED-TMR algorithm described, refines a standardhydrophobicity analysis with a detection of potential termini(`edges', starts and ends) of transmembrane regions. This allowsone both to discard highly hydrophobic regions not delimitedby clear start and end configurations and to confirm putativetransmembrane segments not distinguishable by their hydrophobiccomposition. The accuracy obtained on a test set of 101 non-homologoustransmembrane proteins with reliable topologies compares wellwith that of other popular existing methods. Only a slight decreasein prediction accuracy was observed when the algorithm was appliedto all transmembrane proteins of the SwissProt database (release35). A WWW server running the PRED-TMR algorithm is availableat http://o2.db.uoa.gr/PRED-TMR/     

Recombinant calpain II: improved expression systems and production of a C105A active-site mutant for crystallography

The bacterial production of recombinant rat calpain II has beenimproved greatly by the use of two compatible plasmids for thetwo subunits. The calpain small subunit C-terminal fragment(21 kDa) was expressed from a new A15-based vector created bycloning T7 control elements into pACYC177. This vector is compatiblewith the ColElbased pET-24d(+) vector containing the calpainlarge subunit, and the yield of calpain activity was increasedat least 16-fold by co-expression from these two vectors. Ahigh level of activity was also obtained from a bicistronicconstruct containing both subunit cDNAs under the control ofone T7 promoter. The addition of a C-terminal His-tag to thelarge subunit simplified purification without affecting subunitassociation or enzyme activity. The active-site cysteine 105was mutated to alanine, causing complete loss of activity. Theyield of purified C105A-calpain II (80 + 21 kDa) dimer followingthree column chromatography steps was 10 mg/l of cell culture.This provides a purified calpain, stable to autolysis and oxidation,which is likely to facilitate crystallization in both the presenceand absence of calcium.     

Effects of hydrophobic amino acid substitution in Pleurotus ostreatus proteinase A inhibitor 1 on its structure and functions as protease inhibitor and intramolecular chaperone

We previously demonstrated that Pleurotus ostreatus proteinase A inhibitor 1 (POIA1) could function as an intramolecular chaperone of subtilisin BPN', as in the case of the propeptide of subtilisin BPN', and that its Phe44 --> Ala mutant, which lost its tertiary structure, could not assist the refolding of subtilisin BPN'. In this study, we examined the effects of hydrophobic amino acid substitutions at other sites and substitutions of Phe44 with other hydrophobic residues on the structure and functions of POIA1. These mutations were introduced into POIA1cm that had been obtained by the substitution of the C-terminal six residues of POIA1 with those of the propeptide of subtilisin BPN'. When Ile32 or Ile64 was substituted with Ala, the tertiary structure of the resultant mutant was markedly destroyed, and the activities as a protease inhibitor and an intramolecular chaperone were significantly lowered. Among the position 44 mutants, the Phe44 --> Val mutant was a much less effective intramolecular chaperone with conversion to a digestible inhibitor, possibly owing to destruction of the tertiary structure. On the other hand, the Phe44 --> Leu or Ile mutant maintained its tertiary structure, and hence could function as a more effective intramolecular chaperone than the Phe44 --> Val mutant. Furthermore, since the Phe44 --> Leu mutant was a more susceptible inhibitor than POIA1cm, the halo formed around a colony of Bacillus cells transformed with a plasmid encoding this mutant was larger than others. These results clearly show the close relationship between the tertiary structure and functions of POIA1 as a protease inhibitor and an intramolecular chaperone, and that a combination of such inhibitory properties and intramolecular chaperone activity of POIA1 might affect the diameter of the halo formed around Bacillus colonies in vivo.