An adaptive mutation in adenylate kinase that increases organismal fitness is linked to stability-activity trade-offs

Protein function is a balance between activity and stability. However, the relevance of stability-activity trade-offs for protein evolution and their impact on organismal fitness have been difficult to determine. Previously, we have linked organismal survival at increasing temperatures to adaptive changes to a single protein sequence through allelic replacement of an essential gene, adenylate kinase (adk), in a thermophile. In vivo continuous evolution of the temperature-sensitive thermophile has shown that the first step toward increased organismal fitness is mutation of glutamine-199 to arginine in the mesophilic enzyme (AKsub Q199R). Here, we show that although substitution of Arg-199 did confer a modest increase in stability (0.6 kcal mol(-1)at 20 degrees C; DeltaT(m) = 3.0 degrees C), it is a large change in the activity profile of the enzyme that is responsible for its exceptional robustness during the earlier experimental evolution study. Kinetic studies of AKsub Q199R show that it has a strong loss of enzymatic activity (>50%) at lower temperatures (20-45 degrees C) and a subsequent increase at elevated temperatures. The stability-activity trade-off observed for AKsub Q199R was linked to the rigidification of the overall structure through stabilization of a polypeptide loop containing Arg-199 that is part of the ATP-binding site of the enzyme. Structural analysis revealed the formation of new ionic interactions facilitated by Arg-199. Our results suggest that stability-activity trade-offs are employed readily as an evolutionary strategy during natural selection to increase organismal fitness.     

Structure prediction of the EcoRV DNA methyltransferase based on mutant profiling, secondary structure analysis, comparison with known structures of methyltransferases and isolation of catalytically inactive single mutants

The EcoRV DNA methyltransferase (M·EcoRV) is an -adeninemethyltransferase. We have used two different programs to predictthe secondary structure of M·EcoRV. The resulting consensusprediction was tested by a mutant profiling analysis. 29 neutralmutations of M·EcoRV were generated by five cycles ofrandom mutagenesis and selection for active variants to increasethe reliability of the prediction and to get a secondary structureprediction for some ambiguously predicted regions. The predictedconsensus secondary structure elements could be aligned to thecommon topology of the structures of the catalytic domains ofM·HhaI and M·TaqI. In a complementary approachwe have isolated nine catalytically inactive single mutants.Five of these mutants contain an amino acid exchange withinthe catalytic domain of M·EcoRV (Val20-Ala, Lys81Arg,Cys192Arg, Asp193Gly, Trp231Arg). The Trp231Arg mutant bindsDNA similarly to wild-type M·EcoRV, but is catalyticallyinactive. Hence this mutant behaves like a bona fide activesite mutant. According to the structure prediction, Trp231 islocated in a loop at the putative active site of M·EcoRV.The other inactive mutants were insoluble. They contain aminoacid exchanges within the conserved amino acid motifs X, IIIor IV in M·EcoRV confirming the importance of these regions.     

The evolutionary landscape of functional model proteins

To study the distinct influences of structure and function onevolution, we propose a minimalist model for proteins with bindingpockets, called functional model proteins, based on a shifted-HPmodel on a two-dimensional square lattice. These model proteinsare not maximally compact and contain an empty lattice sitesurrounded by at least three nearest neighbors, thus providinga binding pocket. Functional model proteins possess a uniquenative state, cooperative folding and tolerance to mutation.Due to the explicit functionality in these models (by design),we have been able to explore their fitness or evolutionary landscapes,as characterized by the size and distribution of homologousfamilies and by the complexity of the inter-relatedness of thefunctional model proteins. Mindful that these minimalist modelsare highly idealized and two-dimensional, functional model proteinsshould nevertheless provide a useful means for exploring theconstraints of maintaining structure and function on the evolutionof proteins.     

Analysis and prediction of the location of catalytic residues in enzymes

The catalytic residues of an enzyme are defined as the aminoacids directly involved in chemical catalysis. They mainly actas a general acid–base, electrophilic or nucleophiliccatalyst or they polarize and stabilize the transition state.An analysis of the structural features of 36 catalytic residuesin 17 enzymes of known structure and with defined mechanismis reported. Residues that bind metal ions (Zn² and Cu²) areconsidered separately. The features examined are: residue type,location in secondary structure, separation between the residues,accessibility to solvent, intra-protein electrostatic interactions,mobility as evaluated from crystallographic temperature factors,polarity of the environment and the sequence conservation betweenhomologous enzymes of residues that were sequentially or spatiallyclose to the catalytic residue. In general the environment ofcatalytic residues is similar to that of polar side chains thathave low accessibility to solvent. Two algorithms have beendeveloped to identify probable catalytic residues. Scanningan alignment of homologous enzyme sequences for peaks of sequenceconservation identifies 13 out of the 16 catalytic residueswith 50 residues overpredicted. When the conservation of thespatially close residues is used instead, a different set of13 residues are identified with 47 residues overpredicted. Acombination of the two algorithms identifies 11 residues with36 residues overpredicted.     

Biased mutation-assembling: an efficient method for rapid directed evolution through simultaneous mutation accumulation

We have developed an efficient optimization technique, 'biased mutation-assembling', for improving protein properties such as thermostability. In this strategy, a mutant library is constructed using the overlap extension polymerase chain reaction technique with DNA fragments from wild-type and phenotypically advantageous mutant genes, in which the number of mutations assembled in the wild-type gene is stochastically controlled by the mixing ratio of the mutant DNA fragments to wild-type fragments. A high mixing ratio results in a mutant composition biased to favor multiple-point mutants. We applied this strategy to improve the thermostability of prolyl endopeptidase from Flavobacterium meningosepticum as a case study and found that the proportion of thermostable mutants in a library increased as the mixing ratio was increased. If the proportion of thermostable mutants increases, the screening effort needed to find them should be reduced. Indeed, we isolated a mutant with a 1200-fold longer activity half-life at 60 degrees C than that of wild-type prolyl endopeptidase after screening only 2000 mutants from a library prepared with a high mixing ratio. Our results indicate that an aggressive accumulation of advantageous mutations leads to an increase in the quality of the mutant library and a reduction in the screening effort required to find superior mutants.     

Identification of important functional environs in protein tertiary structures from the analysis of residue variation in 3-D: application to cytochromes c and carboxypeptidases A and B

A simple methodology is described to apply to aligned proteinsequence sets for which at least one representative 3-D C structureis known. The evolutionary variation observed at each residueposition in the sequence alignment is qualified by taking intoaccount the residue variation that has occurred at other positionslocated within 7 A (according to the probable chain fold). Thisexpresses the evolutionary behaviour of any residue positionin the more appropriate context of its immediate surroundingsand distinguishes between invariant residues on the basis ofthe variation of their environment. The highest mechanisticsignificance is attached to conserved residues in conservedsurroundings, but the quantitative nature of the analysis meansthat all residue vicinities can be ranked and merged accordingto the degree of conservation that they exhibit and the residuepositions that comprise them. Therefore, with the aid of thechain fold, contour maps can be constructed that show gradedfoci of evolutionary conservation in the underlying superstructureof the protein type, and the irregular shapes and extents oflarge conserved areas. To test the methodology, it was appliedto cytochromes c and the carboxypeptidases A and B.     

Understanding protein lids: structural analysis of active hinge mutants in triosephosphate isomerase

The conformational switch from open to closed of the flexible loop 6 of triosephosphate isomerase (TIM) is essential for the catalytic properties of TIM. Using a directed evolution approach, active variants of chicken TIM with a mutated C-terminal hinge tripeptide of loop 6 have been generated (Sun,J. and Sampson,N.S., Biochemistry, 1999, 38, 11474-11481). In chicken TIM, the wild-type C-terminal hinge tripeptide is KTA. Detailed enzymological characterization of six variants showed that some of these (LWA, NPN, YSL, KTK) have decreased catalytic efficiency, whereas others (KVA, NSS) are essentially identical with wild-type. The structural characterization of these six variants is reported. No significant structural differences compared with the wild-type are found for KVA, NSS and LWA, but substantial structural adaptations are seen for NPN, YSL and KTK. These structural differences can be understood from the buried position of the alanine side chain in the C-hinge position 3 in the open conformation of wild-type loop 6. Replacement of this alanine with a bulky side chain causes the closed conformation to be favored, which correlates with the decreased catalytic efficiency of these variants. The structural context of loop 6 and loop 7 and their sequence conservation in 133 wild-type sequences is also discussed.     

Natural history as a predictor of protein evolvability

Natural selection generally produces specific and efficient enzymes. In contrast, directed evolution experiments usually produce enzyme variants with broadened substrate specificity or enhanced catalytic promiscuity. Some proteins may be more evolvable than others, but few workers consider this problem when choosing starting points for laboratory evolution. Here, we review the variables associated with enzyme evolvability, namely promiscuity and mutational robustness. We present a qualitative model of adaptive evolution and recommend that protein engineers exploit their knowledge of natural history to identify evolvable wild-type proteins. Three examples of 'generalist' proteins that evolved in the laboratory into 'specialists' are described to illustrate the practical utility of this point.     

Evolutionary divergence and conservation of trypsin

The trypsin sequences currently available in the data bankshave been collected and aligned using first the amino acid sequencehomology and, subsequently, the superposed crystal structuresof trypsins from the cow, the bacterium Streptomyces griseusand the fungus Fusarium oxysporum. The phylogenetic tree constructedaccording to this multiple alignment is consistent with a continuousevolutionary divergence of trypsin from a common ancestor ofboth prokaryotes and eukaryotes. Comparison of crystal structuresreveals a strict conservation of secondary structure. Similarly,in the alignment of all the sequences, insertions and deletionsoccur only in regions corresponding to loops between the secondarystructure elements in the known crystal structures. The conservedresidues cluster around the active site. Almost all conservedresidues can be associated with one of the basic functionalfeatures of the protein: zymogen activation, catalysis and substratespecificity. In contrast, the residues of the hydrophobic coreof the protein and the calcium ion binding sites are generallynot conserved. The conserved features of trypsin and the natureof the conservation are discussed In detail     

Incorporating Synthetic Oligonucleotides via Gene Reassembly (ISOR): a versatile tool for generating targeted libraries

The directed evolution of proteins has benefited greatly from site-specific methods of diversification such as saturation mutagenesis. These techniques target diversity to a number of chosen positions that are usually non-contiguous in the protein's primary structure. However, the number of targeted positions can be large, thus leading to impractically large library size, wherein almost all library variants are inactive and the likelihood of selecting desirable properties is extremely small. We describe a versatile combinatorial method for the partial diversification of large sets of residues. Our library oligonucleotides comprise randomized codons that are flanked by wild-type sequences. Adding these oligonucleotides to an assembly PCR of wild-type gene fragments incorporates the randomized cassettes, at their target sites, into the reassembled gene. Varying the oligonucleotides concentration resulted in library variants that carry a different average number of mutated positions that comprise a random subset of the entire set of diversified codons. This method, dubbed Incorporating Synthetic Oligos via Gene Reassembly (ISOR), was used to create libraries of a cytosine-C5 methyltransferase wherein 45 individual positions were randomized. One library, containing an average of 5.6 mutated residues per gene, was selected, and mutants with wild-type-like activities isolated. We also created libraries of serum paraoxonase PON1 harboring insertions and deletions (indels) in various areas surrounding the active site. Screening these libraries yielded a range of mutants with altered substrate specificities and indicated that certain regions of this enzyme have a surprisingly high tolerance to indels.     

Can three-dimensional contacts in protein structures be predicted by analysis of correlated mutations?

A method has been developed to detect pairs of positions withcorrelated mutations in protein multiple sequence alignments.The method is based on reconstruction of the phylogenetic treefor a set of sequences and statistical analysis of the distributionof mutations in the branches of the tree. The database of homology-derivedprotein structures (HSSP) is used as the source of multiplesequence alignments for proteins of known three-dimensionalstructure. We analyse pairs of positions with correlated mutationsin 67 protein families and show quantitatively that the presenceof such positions is a typical feature of protein families.A significant but weak tendency is observed for correlated residuepairs to be close in the three-dimensional structure. With furtherimprovements, methods of this type may be useful for the predictionof residue-residue contacts and subsequent prediction of proteinstructure using distance geometry algorithms. In conclusion,we suggest a new experimental approach to protein structuredetermination in which selection of functional mutants afterrandom mutagenesis and analysis of correlated mutations providesufficient proximity constraints for calculation of the proteinfold     

Potential of genetic algorithms in protein folding and protein engineering simulations

Genetic algorithms are very efficient search mechanisms whichmutate, recombine and select amongst tentative solutions toa problem until a near optimal one is achieved. We introducethem as a new tool to study proteins. The identification andmotivation for different fitness functions is discussed. Theevolution of the zinc finger sequence motif from a random startis modelled. User specified changes of the repressor structurewere simulated and critical sites and exchanges for mutagenesisidentified. Vast conformational spaces are efficiently searchedas illustrated by the ab initio folding of a model protein ofa four ß strand bundle. The genetic algorithm simulationwhich mimicked important folding constraints as overall hydrophobicpackaging and a propensity of the betaphilic residues for transpositions achieved a unique fold. Cooperativity in the ßstrand regions and a length of 3–5 for the interconnectingloops was critical. Specific interaction sites were considerablyless effective in driving the fold.     

Functional analysis of organophosphorus hydrolase variants with high degradation activity towards organophosphate pesticides

Organophosphorus hydrolase (OPH, also known as phosphotriesterase) is a bacterial enzyme that is capable of degrading a wide range of neurotoxic organophosphate nerve agents. Directed evolution has been used to generate one variant (22A11) with up to 25-fold improved hydrolysis of methyl parathion. Surprisingly, this variant also degraded all other substrates (paraoxon, parathion and coumaphos) tested 2- to 10-fold faster. Since only one mutation (H257Y) is directly located in the active site, site-directed mutagenesis and saturation mutagenesis were used to identify the role of the other distal substitutions (A14T, A80V, K185R, H257Y, I274N) on substrate specificity and activity. Sequential site-directed mutagenesis indicated that K185R and I274N are the most important substitutions, leading to an improvement not only in the hydrolysis of methyl parathion but also the overall hydrolysis rate of all other substrates tested. Using structural modeling, these two mutations were shown to favor the formation of hydrogen bonds with nearby residues, resulting in structural changes that could alter the overall substrate hydrolysis.     

An improved prediction of catalytic residues in enzyme structures

The protein databases contain a huge number of function unknown proteins, including many proteins with newly determined 3D structures resulted from the Structural Genomics Projects. To accelerate experiment-based assignment of function, de novo prediction of protein functional sites, like active sites in enzymes, becomes increasingly important. Here, we attempted to improve the prediction of catalytic residues in enzyme structures by seeking and refining different encodings (i.e. residue properties) as well as employing new machine learning algorithms. In particular, considering that catalytic residues can often reveal specific network centrality when representing enzyme structure as a residue contact network, the corresponding measurement (i.e. closeness centrality) was used as one of the most important encodings in our new predictor. Meanwhile, a genetic algorithm integrated neural network (GANN) was also employed. Thanks to the above strategies, our GANN predictor demonstrated a high accuracy of 91.2% in the prediction of catalytic residues based on balanced datasets (i.e. the 1:1 ratio of catalytic to non-catalytic residues). When the GANN method was optimally applied to real enzyme structures, 73.9% of the tested structures had the active site correctly located. Compared with two existing methods, the proposed GANN method also demonstrated a better performance.     

Conservation of residue interactions in a family of Ca-binding proteins

In the TNC family of Ca-binding proteins (calmodulin, parvalbumin,intestinal calcium binding protein and troponin C) {small tilde}70 well-conserved amino acid sequences and six crystal structuresare known. We find a clear correlation between residue contactsin the structures and residue conservation in the sequences:residues with strong sidechain–sidechain contacts in thethree-dimensional structure tend to be the more conserved inthe sequence. This is one way to quantify the intuitive notionof the importance of sidechain interactions for maintainingprotein three-dimensional structure in evolution and may usefullybe taken into account in planning point mutations in proteinengineering.     

A thermostable variant of fructose bisphosphate aldolase constructed by directed evolution also shows increased stability in organic solvents

Thermostable variants of the Class II fructose bisphosphate aldolase have been isolated following four rounds of directed evolution using DNA shuffling of the fda genes from Escherichia coli and Edwardsiella ictaluri. Variants from all four generations of evolution have been purified and characterized. The variants show increased thermostability with no loss of catalytic function at room temperature. The temperature at which 50% of the initial enzyme activity is lost after incubation for 10 min (T50) of the most stable variant, 4-43D6, is increased by 11-12 degrees C over the wild-type enzymes and the half-life of activity at 53 degrees C is increased approximately 190-fold. In addition, variant 4-43D6 shows increased stability to treatment with organic solvents. DNA sequencing of the evolved variants has identified the mutations which have been introduced and which lead to increased thermostability, and the role of the mutations introduced is discussed.     

The prediction of protein contacts from multiple sequence alignments

We have studied the question of how much extra predictive powerthe correlated mutational behaviour of pairs of amino acid residuesseparated along a sequence has concerning the likelihood ofthose residues being in contact in the folded protein. The mutationalbehaviour is deduced from multiple sequence alignments. Ourfindings are that there is, indeed, some valuable informationavailable from this source and that it is sufficient to makea significant improvement in our ability to predict contacts,when compared with earlier methods that do not take into accountthe correlations between the mutations. This improvement isapproximately twice as large as can be obtained by the moreeconomical method of simply averaging pair preferences overthe same sequence alignment. Even when using a method basedon pair preferences, a further significant improvement can bemade by penalizing more variable regions (on the reasonableassumption that invariant residues are relatively more likelyto be in contact), though we have found no way of improvingthe pair preference method to the extent that it matches themethod based on correlated behaviour. Our new method is thoughtto be the best data-based method of contact prediction developedso far, achieving, on average, an improvement over a random(i.e. information-free) prediction of a factor of five whenthe number of contacts predicted is chosen to match the numberthat actually occur.     

Light-chain framework region residue Tyr71 of chimeric B72.3 antibody plays an important role in influencing the TAG72 antigen binding

The crystallographic study of chimeric B72.3 antibody illustratedthat there are three FR side-chain interactions with eitherCDR residue's side chain or main chain. For example, hydrogenbonds are formed between the hydroxyl group of threonine atL5 in FR1 and the guanidinal nitrogen group of arginine at L24in CDR1, between the hydroxyl group of tyrosine at L36 in FR2and the amide nitrogen group of glutamine at L89 in CDR3 andbetween the hydroxyl group of tyrosine at L71 in FR3 and thecarbonyl group of isoleucine at L29 as well as the amide nitrogengroup of serine at L31 in CDR1. Elimination of these hydrogenbonds at these FR positions may affect CDR loop conformations.To confirm these assumptions, we altered these FR residues bysite-directed mutagenesis and determined binding affinitiesof these mutant chimeric antibodies for the TAG72 antigen. Wefound that the substitution of tyrosine by phenylalanine atL71, altering main-chain hydrogen bonds, significantly reducedthe binding affinity for the TAG72 antigen by 23-fold, whereasthe substitution of threonine and tyrosine by alanine and phenylalanineat L5 and L36, eliminating hydrogen bonds to side-chain atoms,did not affect the binding affinity for the TAG72 antigen. Ourresults indicate that the light-chain FR residue tyrosine atL71 of chimeric B72.3 antibody plays an important role in influencingthe TAG72 antigen binding. Our results will thus be of importancewhen the humanized B72.3 antibody is constructed, since thisimportant mouse FR residue tyrosine at L71 must be maintained.     

A method for construction of long randomized open reading frames and polypeptides

A method is presented for construction of randomized open readingframe sequences (ORFs) and gene libraries containing them. Thebuilding blocks for the ORFs were 75 bp long DNA fragments generatedby cloning sequences from a single synthetic oligonucleotidepreparation by bridge mutagenesis. The fragments had the propertythat, regardless of their orientation in the ligated product,the ORF of the construct was maintained. The heterogeneity ofthe ORFs resulted from the random ligation of 2000 differentDNA fragments. The randomized ORFs were cloned downstream fromthe lac promoter in a multicopy plasmid in Escherichia coli.To test the method, a library of 10⁶ clones was constructed.     

The function of the Saccharomyces cerevisiae iso-1-cytochrome c gene is independent of the codon at invariant residue Phe82 when the gene is present on a low-copy-number vector

Phe82 is the most studied invariant residue of cytochrome c.However, the physiological relevance of amino acid substitutionsat this position is unclear because previous studies were eitherperformed in vitro (i.e. using purified protein) or in yeastwhere the gene for the protein is present on a multi-copy vector.Multi-copy vectors yield a level of cytochrome c in yeast thatis greater than the wild-type level. Oligodeoxyribonucleotide-directedmutagenesis was used to change the codon for Phe82 to that ofthe other 19 naturally occurring amino acids as well as theamber stop codon. The alleles are present on a yeast shuttlephagemid containing the CEN6 gene which ensures a vector copynumber of one to two in yeast. All the missense alleles supportgrowth under conditions requiring a functional iso-1-cytochromec. However the F82C, F82P, and F82R variants grow at a significantlylower rate. After selection for function, phagemids were rescuedfrom the transformants and the identity of the mutation verified.It is concluded that all 20 amino acids are capable of supportingfunction. Reasons for the evolutionary invariance of Phe82 arediscussed.