Synthesis of a novel histidine analogue and its efficient incorporation into a protein in vivo

Proteins containing unnatural amino acids have immense potentialin biotechnology and medicine. We prepared several histidineanalogues including a novel histidine analogue, ß-(1,2,3-triazol-4-yl)-DL-alanine.These histidine analogues were assayed for translational activityin histidine-auxotrophic Escherichia coli strain UTH780. Weobserved that several histidine analogues, including our novelhistidine analogue, were efficiently incorporated into the proteinin vivo; however, other analogues were rejected. These resultssuggest that the hydrogen atom at a specific position seriouslyaffects incorporation. Received April 10, 2003; revised June 20, 2003; accepted July 22, 2003.     

A systematic approach for yielding a potential pool of enzymes: practical case for chiral resolution of (R,S)-ketoprofen ethyl ester

A systematic approach for the selection of potential biocatalystsfrom a natural source was developed and then a practical applicationwas addressed. The approach that involves systematically combinedconventional screening methods and current tools comprises thefollowing consecutive steps: strain enrichment for activityscreening, identification of positive strains, choosing wholegenome-sequenced strains as candidates, gathering informationabout responsible enzymes, bioinformatic analyses and gene mining,probing genetic molecules and then functional expression. Thetarget compound (R,S)-ketoprofen ethyl ester was to be resolvedinto an enantiomer, and a potential esterase from Pseudomonasfluorescens KCTC 1767 was prepared by the proposed procedure.The enzyme had a high activity and also strict selectivity forthe enantiomer (S)-ketoprofen and was suitable therefore asa biocatalyst for practical use. The result achieved by thecombined approach could not easily be obtained using other approacheswith typical procedures. Hence the approach proposed here shouldbe of considerable use for the screening of potential enzymes,particularly for enzymes with desired activity to unnaturalsubstrates, from conditionally expressed and/or repressed proteinsthat are distributed widely in natural pools under normal conditions. Received July 4, 2002; revised January 20, 2003; accepted April 4, 2003.     

Role of tryptophan 121 in the soluble CuA domain of cytochrome c oxidase: structure and electron transfer studies

To investigate the contribution of tryptophan-121 (Trp121) residueto the structure and function of soluble Cu_A domain of cytochromec oxidase, three mutant proteins, Trp121Tyr, Trp121Leu and Trp121-deletedmutant of the soluble domain of Paracoccus versutus cytochromec oxidase, were constructed and expressed in Escherichia coliBL21 (DE3). Optical spectral studies showed that both the coordinationstructure of the Cu_A center and the secondary structure of theprotein were changed significantly in the Leu substitution anddeletion mutants of Trp121. Their electron transfer activitywith cytochrome c was inhibited severely, as shown in stopped-flowkinetic studies. However, the Cu_A center can be reconstructedin the Trp121Tyr mutant although its stability decreases comparedwith the wild-type protein. This mutant keeps the same secondarystructure as the wild-type protein, but can only transfer electronswith cytochrome c at a rate of one-seventh-fold. Based on theinformation on the structure, we also investigated and analyzedthe possible factors that affect electron transfer. It appearsthat the aromatic ring, the size of the side chain and the hydrogenbonding ability of the Trp121 are crucial to the structure andfunction of the soluble Cu_A domain. Received September 11, 2002; revised February 26, 2003; accepted May 27, 2003.     

Vicinal disulfide turns

The formation of a disulfide bond between adjacent cysteineresidues is accompanied by the formation of a tight turn ofthe protein backbone. In nearly 90% of the structures analyzeda type VIII turn was found. The peptide bond between the twocysteines is in a distorted trans conformation, the omega torsionangle ranges from 159 to –133°, with an average valueof 171°. The constrained nature of the vicinal disulfideturn and the pronounced difference observed between the oxidizedand reduced states, suggests that vicinal disulfides may beemployed as a ‘redox-activated’ conformational switch. Received December 16, 2002; revised June 30, 2003; accepted July 30, 2003.     

Immobilized oxidoreductase as an additive for refolding inclusion bodies: application to antibody fragments

Three foldases—the apical domain of GroEL (mini-chaperone)and two oxidoreductases (DsbA and DsbC) from Escherichia coli—werestudied in refolding a protein with immunoglobulin fold (immunoglobulin-foldedprotein) that had been produced as inclusion bodies in E.coli.The foldases promoted the refolding of single-chain antibodyfragments from denaturant-solubilized and reduced inclusionbodies in vitro, and also effectively functioned as alternativesfor labilizing agent and oxidizing reagent in the stepwise dialysissystem. Immobilization of the oxidoreductases enhanced refoldingand recovery of functional single-chain antibody in the dialysissystem, suggesting that immobilized oxidoreductases can be usedas an effective additive for refolding immunoglobulin-foldedproteins in vitro. Received April 7, 2003; revised June 5, 2003; accepted June 6, 2003.     

PROSPECT II: protein structure prediction program for genome-scale applications

A new method for fold recognition is developed and added tothe general protein structure prediction package PROSPECT (http://compbio.ornl.gov/PROSPECT/).The new method (PROSPECT II) has four key features. (i) We havedeveloped an efficient way to utilize the evolutionary informationfor evaluating the threading potentials including singletonand pairwise energies. (ii) We have developed a two-stage threadingstrategy: (a) threading using dynamic programming without consideringthe pairwise energy and (b) fold recognition considering allthe energy terms, including the pairwise energy calculated fromthe dynamic programming threading alignments. (iii) We havedeveloped a combined z-score scheme for fold recognition, whichtakes into consideration the z-scores of each energy term. (iv)Based on the z-scores, we have developed a confidence index,which measures the reliability of a prediction and a possiblestructure–function relationship based on a statisticalanalysis of a large data set consisting of threadings of 600query proteins against the entire FSSP templates. Tests on severalbenchmark sets indicate that the evolutionary information andother new features of PROSPECT II greatly improve the alignmentaccuracy. We also demonstrate that the performance of PROSPECTII on fold recognition is significantly better than any othermethod available at all levels of similarity. Improvement inthe sensitivity of the fold recognition, especially at the superfamilyand fold levels, makes PROSPECT II a reliable and fully automatedprotein structure and function prediction program for genome-scaleapplications. Received March 20, 2003; revised June 28, 2003; accepted July 8, 2003.     

Prediction of protein secondary structure with a reliability score estimated by local sequence clustering

Most algorithms for protein secondary structure prediction arebased on machine learning techniques, e.g. neural networks.Good architectures and learning methods have improved the performancecontinuously. The introduction of profile methods, e.g. PSI-BLAST,has been a major breakthrough in increasing the prediction accuracyto close to 80%. In this paper, a brute-force algorithm is proposedand the reliability of each prediction is estimated by a z-scorebased on local sequence clustering. This algorithm is intendedto perform well for those secondary structures in a proteinwhose formation is mainly dominated by the neighboring sequencesand short-range interactions. A reliability z-score has beendefined to estimate the goodness of a putative cluster foundfor a query sequence in a database. The database for predictionwas constructed by experimentally determined, non-redundantprotein structures with <25% sequence homology, a list maintainedby PDBSELECT. Our test results have shown that this new algorithm,belonging to what is known as nearest neighbor methods, performedvery well within the expectation of previous methods and thatthe reliability z-score as defined was correlated with the reliabilityof prediction. This led to the possibility of making very accuratepredictions for a few selected residues in a protein with anaccuracy measure of Q₃ > 80%. The further development ofthis algorithm, and a nucleation mechanism for protein foldingare suggested. Received March 27, 2003; revised June 30, 2003; accepted August 22, 2003.     

A long insertion reverts the functional effect of a substitution in acetylcholinesterase

Proteins are thought to undertake single substitutions, deletionsand insertions to explore the fitness landscape. Nevertheless,the ways in which these different kind of mutations act togetherto alter a protein phenotype remain poorly described. We introducedincrementally the single substitution W290A and a 26 amino acidlong insertion at the 297 location in the Nippostrongylus brasiliensisacetylcholinesterase B sequence and analysed in vitro the inducedchanges in the hydrolysis rate of three hemi-substrates: pirimicarb,paraoxon methyl and omethoate. The substitution decreased thehydrolysis rate of the three hemi-substrates. The insertiondid not influence this kinetic alteration induced by the substitutionfor the former hemi-substrate, but reverted it for the two others.These results show that two different kinds of mutations caninteract together to influence the direction of a protein’sadaptative walk on the fitness landscape. Received January 28, 2003; revised April 25, 2003; accepted June 6, 2003.     

Inhibition of the CD28-CD80 co-stimulation signal by a CD28-binding affibody ligand developed by combinatorial protein engineering

CD28 is one of the key molecules for co-stimulatory signallingin T cells. Here, novel ligands (affibodies) showing selectivebinding to human CD28 (hCD28) have been selected by phage displaytechnology from a protein library constructed through combinatorialmutagenesis of a 58-residue three-helix bundle domain derivedfrom staphylococcal protein A. Analysis of selected affibodiesshowed a marked sequence homology and biosensor analyses showedthat all investigated affibodies bound to hCD28 with micromolaraffinities (K_D). No cross-reactivity towards the related proteinhuman CTLA-4 could be observed. This lack of cross-reactivityto hCTLA-4 suggests that the recognition site on hCD28 for theaffibodies resides outside the conserved MYPPPYY motif. Theapparent binding affinity for hCD28 could be improved throughfusion to an Fc fragment fusion partner, resulting in a divalentpresentation of the affibody ligand. For the majority of selectedanti-CD28 affibodies, in co-culture experiments involving JurkatT-cells and CHO cell lines transfected to express human CD80(hCD80) or LFA-3 (hLFA-3) on the cell surface, respectively,pre-incubation of Jurkat cells with affibodies resulted in inhibitionof IL-2 production when they were co-cultured with CHO (hCD80⁺)cells, but not with CHO (hLFA-3⁺) cells. For one affibody variantdenoted Z_CD28:5 a clear concentration-dependent inhibition wasseen, indicating that this affibody binds hCD28 and specificallyinterferes in the interaction between hCD28 and hCD80. Received March 11, 2003; revised June 30, 2003; accepted July 30, 2003.     

Simple consensus procedures are effective and sufficient in secondary structure prediction

We have analyzed the performance of majority voting on minimalcombination sets of three state-of-the-art secondary structureprediction methods in order to obtain a consensus prediction.Using three large benchmark sets from the EVA server, our resultsshow a significant improvement in the average Q₃ predictionaccuracy of up to 1.5 percentage points by consensus formation.The application of an additional trivial filtering procedurefor predicted secondary structure elements that are too short,does not significantly affect the prediction accuracy. Our analysisalso provides valuable insight into the similarity of the resultsof the prediction methods that we combine as well as the higherconfidence in consistently predicted secondary structure. Received March 7, 2003; revised May 24, 2003; accepted June 6, 2003.     

The structural roles of conserved Pro196, Pro197 and His199 in the mechanism of thymidylate synthase

We generated replacement sets for three highly conserved residues,Pro196, Pro197 and His199, that flank the catalytic nucleophile,Cys198. Pro196 and Pro197 have restricted mobility that couldbe important for the structural transitions known to be essentialfor activity. To test this hypothesis we obtained and characterized13 amino acid substitutions for Pro196, 14 for Pro197 and 14for His199. All of the Pro196 and Pro197 variants, except P197R,and four of the His199 variants complemented TS-deficient Escherichiacoli cells, indicating they had at least 1% of wild-type activity.For all His199 mutations, k_cat/K_m for substrate and cofactordecreased more than 40-fold, suggesting that the conserved hydrogenbond network co-ordinated by His199 is important for catalysis.Pro196 can be substituted with small hydrophilic residues withlittle loss in k_cat, but 15- to 23-fold increases in K_m^dUMP.Small hydrophobic substitutions for Pro197 were most active,and the most conservative mutant, P197A, had only a 5-fold lowerk_cat/K_m^dUMP than wild-type TS. Several Pro196 and Pro197 variantswere temperature sensitive. The small effects of Pro196 or Pro197mutations on enzyme kinetics suggest that the conformationalrestrictions encoded by the Pro–Pro sequence are largelymaintained when either member of the pair is mutated. Received February 24, 2003; revised June 19, 2003; accepted June 20, 2003.     

Structure-based substitutions for increased solubility of a designed protein

Manipulation of protein solubility is important for many aspectsof protein design and engineering. Previously, we designed aseries of consensus ankyrin repeat proteins containing one,two, three and four identical repeats (1ANK, 2ANK, 3ANK and4ANK). These proteins, particularly 4ANK, are intended for useas a universal scaffold on which specific binding sites canbe constructed. Despite being well folded and extremely stable,4ANK is soluble only under acidic conditions. Designing interactionswith naturally occurring proteins requires the designed proteinto be soluble at physiological pH. Substitution of six leucineswith arginine on exposed hydrophobic patches on the surfaceof 4ANK resulted in increased solubility over a large pH range.Study of the pH dependence of stability demonstrated that 4ANKis one of the most stable ankyrin repeat proteins known. Inaddition, analogous leucine to arginine substitutions on thesurface of 2ANK allowed the partially folded protein to assumea fully folded conformation. Our studies indicate that replacementof surface-exposed hydrophobic residues with positively chargedresidues can significantly improve protein solubility at physiologicalpH. Received June 23, 2003; revised August 22, 2003; accepted August 28, 2003.     

Optimizing the search algorithm for protein engineering by directed evolution

An in silico protein model based on the Kauffman NK-landscape,where N is the number of variable positions in a protein andK is the degree of coupling between variable positions, wasused to compare alternative search strategies for directed evolution.A simple genetic algorithm (GA) was used to model the performanceof a standard DNA shuffling protocol. The search effectivenessof the GA was compared to that of a statistical approach calledthe protein sequence activity relationship (ProSAR) algorithm,which consists of two steps: model building and library design.A number of parameters were investigated and found to be importantfor the comparison, including the value of K, the screeningsize, the system noise and the number of replicates. The statisticalmodel was found to accurately predict the measured activitiesfor small values of the coupling between amino acids, K 1.The ProSAR strategy was found to perform well for small to moderatevalues of coupling, 0 K 3, and was found to be robust to noise.Some implications for protein engineering are discussed. Received January 2, 2003; revised May 13, 2003; accepted June 19, 2003.     

Structure-oriented rational design of chymotrypsin inhibitor models

Three peptides modelling a highly potent, 35-residue chymotrypsininhibitor (Schistocerca gregaria chymotrypsin inhibitor) weredesigned and synthesized by convergent peptide synthesis. Foreach model peptide, the inhibitory constant (K_i) on chymotrypsinand the solution structure were determined. In addition, moleculardynamics calculations were performed for all of them. Two modelscontaining approximately half of the parent inhibitor (17 of35 residues) were designed and subsequently found to have nosubstantial inhibitory activity (K_i values in the mM range).The third model composed of 24 amino acid residues proved tobe an effective (K_i 10^–7) inhibitor of bovine chymotrypsin.Both the solution structure properties determined by NMR spectroscopyand the dynamic behaviour of the latter model system are comparableto the native inhibitor. In contrast, the structure and dynamicsof the first two related model peptides show characteristicdifferences. We suggest that the conformation and flexibilityof the modelled protease inhibitor are crucial for its biologicalefficiency. Moreover, the structural and dynamic features ofthe binding loop (28–33) and those of the rest of themolecule appear to be interdependent. Most importantly, thesestructural characteristics can be rationally modified, at leastpartially, by peptide design. Received March 7, 2003; revised August 25, 2003; accepted August 26, 2003.     

Specificity grafting of human antibody frameworks selected from a phage display library: generation of a highly stable humanized anti-CD22 single-chain Fv fragment

A prerequisite for the enrichment of antibodies screened fromphage display libraries is their stable expression on a phageduring multiple selection rounds. Thus, if stringent panningprocedures are employed, selection is simultaneously drivenby antigen affinity, stability and solubility. To take advantageof robust pre-selected scaffolds of such molecules, we graftedsingle-chain Fv (scFv) antibodies, previously isolated froma human phage display library after multiple rounds of in vitropanning on tumor cells, with the specificity of the clinicallyestablished murine monoclonal anti-CD22 antibody RFB4. We showthat a panel of grafted scFvs retained the specificity of themurine monoclonal antibody, bound to the target antigen withhigh affinity (6.4–9.6 nM), and exhibited exceptionalbiophysical stability with retention of 89–93% of theinitial binding activity after 6 days of incubation in humanserum at 37°C. Selection of stable human scaffolds withhigh sequence identity to both the human germline and the rodentframeworks required only a small number of murine residues tobe retained within the human frameworks in order to maintainthe structural integrity of the antigen binding site. We expectthis approach may be applicable for the rapid generation ofhighly stable humanized antibodies with low immunogenic potential. Received June 10, 2003; accepted August 27, 2003.     

Molecular docking of substrates and inhibitors in the catalytic site of CYP6B1, an insect cytochrome P450 monooxygenase

Furanocoumarins represent plant toxins that are used in thetreatment of a variety of skin diseases and are metabolizedby cytochrome P450 monooxygenases (P450s) existing in insectssuch as Papilio polyxenes (the black swallowtail). To elucidatethe active site in the CYP6B1 protein that is the principalP450 existing in this species, we have constructed a homologymodel of it based on sequence and structure alignments withthe bacterial CYP102 protein whose crystal structure has beendefined and with the insect CYP6B4 protein that also metabolizesfuranocoumarins. In the derived CYP6B1 model, Phe116 and His117in SRS1, Phe371 in SRS5 and Phe484 in SRS6 contribute to theformation of a resonant network that stabilizes the P450’scatalytic site and allows for interactions with its furanocoumarinsubstrates. The first two of these residues are absolutely conservedin all members of the insect CYP6B subfamily and the last twoare variable in different members of the CYP6B subfamily. Acombination of theoretical and experimental docking analysesof two substrates (xanthotoxin and bergapten) and two inhibitors(coumarin and pilocarpine) of this P450 provide significantinformation on the positioning of furanocoumarins within thiscatalytic pocket. Molecular replacement models based on theresults of variations at two of these critical amino acids providesupport for our furanocoumarin-docked model and begin to rationalizethe altered substrate reactivities observed in experimentalanalyses. Received December 4, 2002; revised June 8, 2003; accepted June 23, 2003.     

Effect of mutations involving charged residues on the stability of staphylococcal nuclease: a continuum electrostatics study

A continuum electrostatics model is used to calculate the relativestabilities of 117 mutants of staphylococcal nuclease (SNase)involving the mutation of a charged residue to an unchargedresidue. The calculations are based on the crystallographicstructure of the wild-type protein and attempt to take implicitlyinto account the effect of the mutations in the denatured stateby assuming a linear relationship between the free energy changescaused by the mutation in the native and denatured states. Agood correlation (linear correlation coefficient of 0.8) isfound with published experimental relative stabilities of thesemutants. The results suggest that in the case of SNase (i) chargedresidues contribute to the stability of the native state mainlythrough electrostatic interactions, and (ii) native-like electrostaticinteractions may persist in the denatured state. The continuumelectrostatics method is only moderately sensitive to modelparameters and leads to quasi-predictive results for the relativemutant stabilities (error of 2–3 kJ mol^–1 or ofthe order of k_BT), except for mutants in which a charged residueis mutated to glycine. Received March 24, 2003; revised August 11, 2003; accepted September 12, 2003.     

Improving tolerance of Candida antarctica lipase B towards irreversible thermal inactivation through directed evolution

To expand the functionality of lipase B from Candida antarctica(CALB) we have used directed evolution to create CALB mutantswith improved resistance towards irreversible thermal inactivation.Two mutants, 23G5 and 195F1, were generated with over a 20-foldincrease in half-life at 70°C compared with the wild-typeCALB (WT-CALB). The increase in half-life was attributed toa lower propensity of the mutants to aggregate in the unfoldedstate and to an improved refolding. The first generation mutant,23G5, obtained by error-prone PCR, had two amino acid mutations,V210I and A281E. The second generation mutant, 195F1, derivedfrom 23G5 by error-prone PCR, had one additional mutation, V221D.Amino acid substitutions at positions 221 and 281 were determinedto be critical for lipase stability, while the residue at position210 had only a marginal effect. The catalytic efficiency ofthe mutants with p-nitrophenyl butyrate and 6,8-difluoro-4-methylumbelliferyloctanoate was also found to be superior to that of WT-CALB. Received May 8, 2003; revised June 9, 2003; accepted June 23, 2003.     

Stabilization of a chitinase from Serratia marcescens by Gly->Ala and Xxx->Pro mutations

This paper describes attempts to increase the kinetic stabilityof chitinase B from Serratia marcescens (ChiB) by the introductionof semi-automatically designed rigidifying mutations of theGlyAla and XxxPro type. Of 15 single mutants, several displayedsignificant increases in thermal stability, whereas most mutantsshowed minor effects. All mutations with non-marginal effectson stability clustered in a limited, surface-exposed regionof the enzyme, indicating that this region is involved in apartial unfolding process that triggers irreversible thermalinactivation (aggregation). A double mutant containing two stabilizingmutations in this region (G188A, A234P) displayed a 10-foldincrease in half-life at 57°C and a 4.2°C increase inapparent T_m. These results show that entropic stabilizationworks well for ChiB and they pinpoint a region whose unfoldingmay be crucial for the kinetic stability of this enzyme. Received June 18, 2003; revised September 3, 2003; accepted September 12, 2003.