Pseudomonas glumae lipase: increased proteolytic stabifity by protein engineering

The feasibility of stabilizing proteins towards proteolyticdegradation was explored by engineering the primary proteolyticcleavage site(s). This novel approach does not require informationon the 3-D structure of the native enzyme. As a model system,the extracellular lipase of Pseudomonas glumae was chosen, whichis sensitive towards degradation by subtilisin-tvpe proteases.The primary proteolytic cleavage in the lipase appeared to belocated between amino acids serine 153 and histidine 154. SincesubtUisins are known to show a preference towards amino acidresidues surrounding the scissile bond, non-preferred aminoadds were introduced in this area. Two concepts were tested:the introduction of arginine or glutainate residues (chargeconcept) and the introduction of proline residues (proUne concept).Although the mutant Upases produced according to either of theseconcepts were still cleaved in the same area, they showed aconsiderably increased stability towards proteolytic degradation.     

Design and application of stimulus-responsive peptide systems

The ability of peptides and proteins to change conformations in response to external stimuli such as temperature, pH and the presence of specific small molecules is ubiquitous in nature. Exploiting this phenomenon, numerous natural and designed peptides have been used to engineer stimulus-responsive systems with potential applications in important research areas such as biomaterials, nanodevices, biosensors, bioseparations, tissue engineering and drug delivery. This review describes prominent examples of both natural and designed synthetic stimulus-responsive peptide systems. While the future looks bright for stimulus-responsive systems based on natural and rationally engineered peptides, it is expected that the range of stimulants used to manipulate such systems will be significantly broadened through the use of combinatorial protein engineering approaches such as directed evolution. These new proteins and peptides will continue to be employed in exciting and high-impact research areas including bionanotechnology and synthetic biology.     

The consequences of engineering an extra disulfide bond in the Penicillium camembertii mono-and diglyceride specific lipase

The extracellular lipase from Penicillium camembertii has uniquesubstrate specificity restricted to mono- and diglycerides.The enzyme is a member of a homologous family of lipases fromfilamentous fungi. Four of these proteins, from the fungi Rhizomucormiehei, Humicola lanuginosa, Rhizopus delemar and P.camembertii,have had their structures elucidated by X-ray crystallography.In spite of pronounced sequence similarities the enzymes exhibitsignificant differences. For example, the thermo-stability ofthe P.camembertii lipase is considerably lower than that ofthe H.lanuginosa enzyme. Since only the P.camembertii enzymelacks the characteristic long disulfide bridge, correspondingto Cys22–Cys268 in the H.lanuginosa lipase, we have engineeredthis disulfide into the former enzyme in the hope of obtaininga significantly more stable fold. The properties of the doublemutant (Y22C and G269C) were assessed by a variety of biophysicaltechniques. The extra disulfide link was found to increase themelting temperature of the protein from 51 to 63°C. However,no difference is observed under reducing conditions, indicatingan intrinsic instability of the new disulfide. The optimal temperaturefor catalytic activity decreased by 10°C and the optimumpH was shifted by 0.7 units to more acidic.     

Analysis of protein conformational characteristics related to thermostability

The thermal stability of proteins was studied, 195 single aminoacid residue replacements reported elsewhere being analysedfor several protein conformational characteristics: type ofresidue replacement; conservative versus nonconservative substitution;replacement being in a homologous stretch of amino acid residues;change in hydrogen bond, van der Waals and secondary structurepropensities; solvent-accessible versus inaccessible replacement;type of secondary structure involved in the substitution; thephysico-chemical characteristics to which the thermostabilityenhancement can be attributed; and the relationship of the replacementsite to the folding intermediates of the protein, when known.From the above analyses, some general rules arise which suggestwhere amino acid substitutions can be made to enhance proteinthermostability: substitutions are conservative according tothe Dayhoff matrix; mainly occur on conserved stretches of residues;preferentially occur on solvent-accessible residues; maintainor enhance the secondary structure propensity upon substitution;contribute to neutralize the dipole moment of the caps of helicesand strands; and tend to increase the number of potential hydrogenbonding or van der Waals contacts or improve hydrophobic packing.     

Improvement of Fc-erythropoietin structure and pharmacokinetics by modification at a disulfide bond

Erythropoietin (Epo) is a cytokine that controls the production of red blood cells (RBCs). Epo acts continuously on RBC precursors to prevent apoptosis, so it is important to maintain high levels of Epo activity when treating anemic patients. We describe here modified human Epo [Epo(NDS)] with mutations His32Gly, Cys33Pro, Trp88Cys and Pro90Ala that result in the rearrangement of the disulfide bonding pattern from Cys29-Cys33 to Cys29-Cys88 and that, in the context of an Fc-Epo(NDS) fusion protein, lead to significantly improved properties. Fc-Epo was secreted from NS/0 myeloma cells as about 35% high molecular weight aggregates, was unstable upon removal of N-linked oligosaccharides and showed poor pharmacokinetics and little efficacy in mice. In contrast, a corresponding Fc-Epo(NDS) was secreted almost exclusively as a unit dimer, was relatively stable to removal of N-linked oligosaccharides, had much improved pharmacokinetic properties and had a significantly improved effect on RBC production. These results indicate that rearrangement of the disulfide bonding pattern in a therapeutic protein can have a significant effect on pharmacokinetics and, potentially, the dosing schedule of a protein drug.     

Analysis of the reaction mechanism and substrate specificity of haloalkane dehalogenases by sequential and structural comparisons

Haloalkane dehalogenases catalyse environmentally importantdehalogenation reactions. These microbial enzymes representobjects of interest for protein engineering studies, attemptingto improve their catalytic efficiency or broaden their substratespecificity towards environmental pollutants. This paper presentsthe results of a comparative study of haloalkane dehalogenasesoriginating from different organisms. Protein sequences andthe models of tertiary structures of haloalkane dehalogenaseswere compared to investigate the protein fold, reaction mechanismand substrate specificity of these enzymes. Haloalkane dehalogenasescontain the structural motifs of /ß-hydrolases and epoxidaseswithin their sequences. They contain a catalytic triad withtwo different topological arrangements. The presence of a structurallyconserved oxyanion hole suggests the two-step reaction mechanismpreviously described for haloalkane dehalogenase from Xanthobacterautotrophicus GJ10. The differences in substrate specificityof haloalkane dehalogenases originating from different speciesmight be related to the size and geometry of an active siteand its entrance and the efficiency of the transition stateand halide ion stabilization by active site residues. Structurallyconserved motifs identified within the sequences can be usedfor the design of specific primers for the experimental screeningof haloalkane dehalogenases. Those amino acids which were predictedto be functionally important represent possible targets forfuture site-directed mutagenesis experiments.     

Finding a new vaccine in the ricin protein fold

Previous attempts to produce a vaccine for ricin toxin have been hampered by safety concerns arising from residual toxicity and the undesirable aggregation or precipitation caused by exposure of hydrophobic surfaces on the ricin A-chain (RTA) in the absence of its natural B-chain partner. We undertook a structure-based solution to this problem by reversing evolutionary selection on the 'ribosome inactivating protein' fold of RTA to arrive at a non-functional, compacted single-domain scaffold (sequence RTA1-198) for presentation of a specific protective epitope (RTA loop 95-110). An optimized protein based upon our modeling design (RTA1-33/44-198) showed greater resistance to thermal denaturation, less precipitation under physiological conditions and a reduction in toxic activity of at least three orders of magnitude compared with RTA. Most importantly, RTA1-198 or RTA1-33/44-198 protected 100% of vaccinated animals against supra-lethal challenge with aerosolized ricin. We conclude that comparative protein analysis and engineering yielded a superior vaccine by exploiting a component of the toxin that is inherently more stable than is the parent RTA molecule.     

Enhanced dead-end elimination in the search for the global minimum energy conformation of a collection of protein side chains

Although the conformational states of protein side chains canbe described using a library of rotamers, the determinationof the global minimum energy conformation (GMEC) of a largecollection of side chains, given fixed backbone coordinates,represents a challenging combinatorial problem with importantapplications in the field of homology modelling. Recently, wehave developed a theoretical framework, called the dead-endelimination method, which allows us to identify efficientlyrotamers that cannot be members of the GMEC. Such dead-endingrotamers can be iteratively removed from the system under studythereby tracking down the size of the combinatorial problem.Here we present new developments to the dead-end eliminationmethod that allow us to handle larger proteins and more extensiverotamer libraries. These developments encompass (i) a procedureto determine weight factors in the generalized dead-end eliminationtheorem thereby enhancing the elimination of dead-ending rotamersand (ii) a novel strategy, mainly based on logical argumentsderived from the logic pairs theorem, to use dead-ending rotamerpairs in the efficient elimination of single rotamers. Thesedevelopments are illustrated for proteins of various sizes andthe flow of the current method is discussed in detail. The effectivenessof dead-end elimination is increased by two orders of magnitudeas compared with previous work. In addition, it now becomesfeasible to use extremely detailed libraries. We also providean appendix in which the validity of the generalized dead-endcriterion is shown. Finally, perspectives for further applicationswhich may now become within reach are discussed.     

A thermostable enzyme as an experimental platform to study properties of less stable homologues

The structural and functional characterization of proteins is frequently hampered by lack of stability or by insufficient assembly of oligomeric proteins in over-expression systems. Using F(1)-ATPase as a case study, we tackled this problem by introducing function-determining domains from a difficult-to-handle variety of an enzyme into a stable homologue.     

The fuzzy-end elimination theorem: correctly implementing the side chain placement algorithm based on the dead-end elimination theorem

Recently it has been shown that the dead-end elimination theoremis a powerful tool in the search for the global minimum energyconformation (GMEC) of a large collection of protein side chainsgiven known backbone coordinates and a library of allowed sidechain conformational states, also known as rotamers. A sidechain placement algorithm based on this theorem iterativelyapplies this theorem to single as well as to pairs of rotamersleading to the identification of rotamers, single or pairs,that are incompatible with the GMEC and that can thus be qualifiedas ‘dead-ending’. Here we formulate a theorem whichproves that contrary to intuition dead-end rotamer pairs cannot simply be discarded from consideration in the iterativeprocess leading to the further elimination of dead-end rotamers.We refer to this theorem as the fuzzy-end elimination theorem.We also describe how the obtained dead-end rotamer pairs cancontribute to the search for the GMEC in the protein side chainplacement problem. Hence the present work forms a theoreticalbasis for the correct implementation of a side chain placementalgorithm based on the dead-end elimination theorem. In addition,possible future perspectives are presented.     

Directed evolution of Pseudomonas aeruginosa lipase for improved amide-hydrolyzing activity

A lipase from Pseudomonas aeruginosa was subjected to directed molecular evolution for increased amide-hydrolyzing (amidase) activity. A single round of random mutagenesis followed by screening for hydrolytic activity for oleoyl 2-naphthylamide as compared with that for oleoyl 2-naphthyl ester identified five mutants with 1.7-2.0-fold increased relative amidase activities. Three mutational sites (F207S, A213D and F265L) were found to affect the amidase/esterase activity ratios. The combination of these mutations further improved the amidase activity. Active-site titration using a fluorescent phosphonic acid ester allowed the molecular activities for the amide and the ester to be determined for each mutant without purification of the lipase. A double mutant F207S/A213D gave the highest molecular activity of 1.1 min(-1) for the amide, corresponding to a 2-fold increase compared with that of the wild-type lipase. A structural model of the lipase indicated that the mutations occurred at the sites near the surface and remote from the catalytic triad, but close to the calcium binding site. This study is a first step towards understanding why lipases do not hydrolyze amides despite the similarities to serine proteases in the active site structure and the reaction mechanism and towards the preparation of a general acyl transfer catalyst for the biotransformation of amides.     

The role of heat-shock and chaperone proteins in protein folding: possible molecular mechanisms

Recently some heat-shock proteins have been linked to functionsof ‘chaperoning’ protein folding in vivo. Here currentexperimental evidence is reviewed and possible requirementsfor such an activity are discussed. It is proposed that onemode of chaperone action is to actively unfold misfolded orbadly aggregated proteins to a conformation from whkh they couldrefold spontaneously; that improperly folded proteins are recognizedby excessive stretches of solvent-exposed backbone, rather thanby exposed hydrophobic patches; and that the molecular mechanismfor unfolding is either repeated binding and dissociation (‘plucking’)or translocation of the protein backbone through a binding cleft(‘threading’), allowing the threaded chain to refoldspontaneously. The observed hydrolysis of ATP would providethe energy for active unfolding. These hypotheses can be appliedto both monomeric folding and oligomeric assembly and are sufficientlydetailed to be open to directed experimental verification.     

Graphical representation of the salient conformational features of protein residues

A composite plot for depicting in two dimensions the conformationand the secondary structural features of protein residues hasbeen developed. Instead of presenting the exact values of themain- and side-chain torsion angles (, and ₁), it indicatesthe region in the three-dimensional conformational space towhich a residue belongs. Other structural aspects, like thepresence of a cis peptide bond and disulfide linkages, are alsodisplayed. The plot may be used to recognize patterns in thebackbone and side-chain conformation along a polypeptide chainand to compare protein structures derived from X-ray crystallography,NMR spectroscopy or molecular modelling studies and also tohighlight the effect of mutation on structure.     

A single amino acid substitution can restore the solubility of aggregated colicin A mutants in Escherichia coli

Mutants of colicin A have been prepared in which the three tryptophanresidues (Trp86, Trpl30 and Trpl40), localized in the C-terminaldomain of the soluble wild-type protein, have been substitutedby phenylalanine. The Trpl40Phe single mutation had the effectof decreasing the percentage of protein that is expressed asinsoluble aggregates. The created hydrophobic cavity decreasedthe stability of the protein during its folding, resulting inpartial aggregation in the cytoplasm of the Escherichia coli-producingcells. Aggregation was increased when Trpl40 was substitutedby Lys, Leu or Cys, or if the Trpl40 mutation was combined withthe Trp86Phe and/ or Trpl30Phe mutations. A single mutation,Lysll3Phe, however, was able to restore the solubility of theaggregated mutants in vivo. Detailed analysis of the 3-D structureof the C-terminal domain of colicin A suggests that fillingof the hydrophobic cavity is responsible for this effect.     

Complex additivity of the effects of suppressor mutations in differing protein environments

In the -complementation of -galactosidase, a defective ß-galactosidaseprotein interacts with an autologous peptide fragment (-peptide)to restore enzymatic activity. Within a specific site of a defective-peptide we have previously isolated a large number of mutations,many of which suppress the functional defect. The -peptide wasoriginally defective due to both insertional and substitutionalsequence alteration near its N-terminus, which provided an increasein the sensitivity of detection of (suppressor) secondary mutationswhich conferred improved function. We have now studied the effectsof the suppressor mutations when the primary deleterious mutationsare sequentially reversed. This was done in intact ß-galactosidase,as we have shown that mutations in the -peptide have relatedfunctional effects in the whole protein. Evidence was obtainedshowing that the effects of at least some suppressor mutationswere not simply additive when the mutations are placed intothe original wild-type protein environment. One suppressor appearedto function less effectively in the normal environment, whileanother when tested in the same manner functioned at a relativelyincreased level. This failure to show simple additivity maybe attributable to the physical proximity of the original defectivemutations and the introduced suppressors. Nevertheless, evenin such cases it may be feasible to use a defective proteinas a sensitive starting point for the identification of mutationswhich improve the wild-type protein.     

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.     

Detection of internal cavities in globular proteins

We have undertaken a study of internal cavities in five proteinstructure groups, each containing different crystallographicstructure determinations of the same protein, to understandbetter the nature of packing defects in protein tertiary architectures.Our results show that cavity detection and consistency of detectionare highly dependent on probe and cavity size, cavity positionwithin the globular protein and the local ‘quality’(r.m.s. deviation) of structural consistency within the group.The consistency of solvent placement within cavities has alsobeen examined. We provide guidelines for estimating the likelihoodof a given cavity to be an actual packing defect or to be aresult of experimental error.     

Enhancement of protein stability by the combination of point mutations in T4 lysozyme is additive

A number of mutations have been shown previously to stabilizeT4 lysozyme. By combining up to seven such mutations in thesame protein, the melting temperature was incrementally increasedby up to 83°C at pH 5.4 (G = 3.6 kcal/mol). This shows thatit is possible to engineer a protein of enhanced thermostabilityby combining a series of rationally designed point mutations.It is also shown that this stabilization is achieved with onlyminor, localized changes in the structure of the protein. Thisis consistent with the observation that the change in stabilityof each of the multiple mutants is, in each case, additive,i.e. equal to the sum of the stability changes associated withthe constituent single mutants. One of the seven substitutions,Asn116 Asp, changes a residue that participates in substratebinding; not surprisingly, it causes a significant loss in activity.Ignoring this mutation, there is a gradual reduction in activityas successively more mutations are combined.     

Engineering the xenobiotic substrate specificity of maize glutathione S-transferase I

Glutathione S-transferases (GSTs) are a heterogeneous family of enzymes that catalyse the conjugation of glutathione (GSH) to electrophilic sites on a variety of hydrophobic substrates. In the present study three amino acid residues (Trp12, Phe35 and Ile118) of the xenobiotic binding site (H-site) of maize GST I were altered in order to evaluate their contribution to substrate binding and catalysis. These residues are not conserved and hence may affect substrate specificity and/or product dissociation. The results demonstrate that these residues are important structural moieties that modulate an enzyme's catalytic efficiency and specificity. Phe35 and Ile118 also participate in k(cat) regulation by affecting the rate-limiting step of the catalytic reaction. The effect of temperature on the catalytic activity of the wild-type and mutant enzymes was also investigated. Biphasic Arrhenius and Eyring plots for the wild-type enzyme showed an apparent transition temperature at 35 degrees C, which seems to be the result of a change in the rate-limiting step of the catalytic reaction. Thermodynamic analysis of the activity data showed that the activation energy increases at low temperatures, whereas the entropy change seems to be the main determinant that contributes to the rate-limiting step at high temperatures.