Protein engineering. Protein biosynthesis and chemical synthesis.
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Protein Engineering↗
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Mechanisms of protein evolution and their application to protein engineering.
Protein engineering holds great promise for the development of new biosensors, diagnostics, therapeutics, and agents for bioremediation. Despite some remarkable successes in experimental and computational protein design, engineered proteins rarely achieve the efficiency or specificity of natural enzymes. Current protein design methods utilize evolutionary concepts, including mutation, recombination, and selection, but the inability to fully recapitulate the success of natural evolution suggests that some evolutionary principles have not been fully exploited. One aspect of protein engineering that has received little attention is how to select the most promising proteins to serve as templates, or scaffolds, for engineering. Two evolutionary concepts that could provide a rational basis for template selection are the conservation of catalytic mechanisms and functional promiscuity. Knowledge of the catalytic motifs responsible for conserved aspects of catalysis in mechanistically diverse superfamilies could be used to identify promising templates for protein engineering. Second, protein evolution often proceeds through promiscuous intermediates, suggesting that templates which are naturally promiscuous for a target reaction could enhance protein engineering strategies. This review explores these ideas and alternative hypotheses concerning protein evolution and engineering. Future research will determine if application of these principles will lead to a protein engineering methodology governed by predictable rules for designing efficient, novel catalysts.
Binding Sites↗
Characterization of a novel stable biocatalyst obtained by protein engineering.
Protein engineering is a powerful tool for the improvement of the properties of biocatalysts. Previously we have applied protein engineering technologies to obtain an extremely stable variant of the thermolysin-like protease from Bacillus stearothermophilus [Van den Burg, Vriend, Veltman, Venema and Eijsink (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 2056-2060]. This variant is much more resistant to denaturing conditions (temperature and denaturing agents) than the wild-type enzyme. An extensive enzymic characterization was undertaken to explore the suitability of the variant as a biocatalyst at high temperatures. By comparing a range of variants with increasing thermal stabilities we show that the additivity of the mutations is accompanied by an increase in activity at elevated temperatures in accordance with the Arrhenius law. The results suggest that the constructed protease variants could be suitable alternatives to proteases that are currently used industrially. Our studies demonstrate how protein engineering can be exploited to obtain high-performance biocatalysts.
Catalysis↗
Predicting oligonucleotide-directed mutagenesis failures in protein engineering.
Protein engineering uses oligonucleotide-directed mutagenesis to modify DNA sequences through a two-step process of hybridization and enzymatic synthesis. Inefficient reactions confound attempts to introduce mutations, especially for the construction of vast combinatorial protein libraries. This paper applied computational approaches to the problem of inefficient mutagenesis. Several results implicated oligonucleotide annealing to non-target sites, termed 'cross-hybridization', as a significant contributor to mutagenesis reaction failures. Test oligonucleotides demonstrated control over reaction outcomes. A novel cross-hybridization score, quickly computable for any plasmid and oligonucleotide mixture, directly correlated with yields of deleterious mutagenesis side products. Cross-hybridization was confirmed conclusively by partial incorporation of an oligonucleotide at a predicted cross-hybridization site, and by modification of putative template secondary structure to control cross-hybridization. Even in low concentrations, cross-hybridizing species in mixtures poisoned reactions. These results provide a basis for improved mutagenesis efficiencies and increased diversities of cognate protein libraries.
Algorithms↗
[Protein splicing and its application in protein engineering].
Protein splicing, which is an intein mediated posttranslational processing, involves a series of intramolecular cleavage-ligation reactions. Intein is an intervening polypeptide which can catalytic self-cleavage from a pre-protein accompanied by the concomitant joining of the two flanking polypeptides (the extein) through a peptide bond. Protein splicing not only enriches genetic theory of posttranslational processing, but also have wide application prospect.
Exteins↗
Protein engineering of protein kinase A catalytic subunits results in the acquisition of novel inhibitor sensitivity.
Analysis of the role of specific protein kinases in signal transduction networks has relied heavily on ATP analog inhibitors. Currently used agents, however, often do not distinguish between kinase family members. Genetic approaches can also be used to inactivate a specific kinase, but these techniques do not afford the rapid kinetics possible with pharmacological inhibitors. To circumvent this problem, modification of the structure of a particular protein kinase can be performed to engineer a drug-target interaction of choice. We have used this method to create protein kinase A (PKA) catalytic subunits with modifications that confer sensitivity to novel ATP analog inhibitors. Mutation of methionine 120 to alanine or glycine in either the Calpha or Cbeta subunits of PKA induces sensitivity to a series of C-3 derivatized pyrazolo[3,4-d]pyrimidine-based inhibitors. Modification of threonine 183 enhances this inhibitor sensitivity. The IC(50) values in cell culture of the most broadly effective agent, 1-NM, ranged from 25 to 200 nm depending upon the combination of modified amino acids and were significantly higher than the potencies observed with H-89. Despite their high sequence conservation, Cbeta enzymes with inhibitor-sensitive amino acids at position 120 showed a substantial loss of overall catalytic activity when used to induce reporter gene transcription in transfected cells. Conversion of position 46 (lysine to isoleucine) rescued the ability of position 120 mutated Cbeta enzymes to induce gene transcription. Application of this combined genetic and pharmacological approach should allow analysis of the specific roles of PKA isoforms in cell culture and in vivo.
Alanine↗
[Introduction to experiments on genetic engineering. X. Protein engineering].
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Protein Engineering↗
Role of arginine 67 in the stabilization of chymotrypsin inhibitor 2: examination of amide proton exchange rates and denaturation thermodynamics of an engineered protein.
We have examined the contribution to protein stability of an interaction involving a charged hydrogen bond from an arginyl side chain (Arg67) in the serine proteinase inhibitor chymotrypsin inhibitor 2 (CI-2), by replacing this side chain with an alanyl residue by protein engineering. Using nuclear magnetic resonance spectroscopy (NMR), we have examined the effect of this mutation on the hydrogen-deuterium exchange rates of several backbone amide protons in the native and engineered proteins at 50 degrees C. These exchange rates provide a localized probe at multiple discrete sites throughout the protein and from comparison of native and mutant exchange rates allow calculation of the difference in free energy of exchange (delta delta Gex) resulting from the mutation. The results show that for the majority of amides observed this mutation results in delta delta Gex of ca. 1.7 kcal mol-1 over the whole CI-2 molecule. However, for two relatively exposed amide protons the exchange rates are found to be far less perturbed, implying that local unfolding mechanisms predominate for these protons. Direct measurement of the stability of both proteins to denaturation by guanidinum hydrochloride shows that the interaction contributes 1.4 kcal mol-1 to the stability of the molecule. This value is comparable to those obtained from the NMR exchange measurements and indicates that the exchange processes reflect the differences in stability between the native and mutant proteins.(ABSTRACT TRUNCATED AT 250 WORDS)
Arginine↗
Engineered protein function by selective amino acid diversification.
Almost all protein engineering methods rely upon making changes to naturally occurring proteins that already possess some of the desired properties. This will probably remain the case as long as we lack a complete understanding of the way that an amino acid sequence gives rise to a protein with a precisely defined biological function. Common to all methods for altering an existing protein is the selection of a subset of amino acids in the protein for variation and a choice of which substitutions to make at each position. Variants are then tested empirically and further variants are created based upon their performance. Differences between protein engineering methods are the ways in which amino acids are chosen for variation, the protocols followed for creating the variants, and how information regarding variant properties is used in creating subsequent variants. In this article, we describe these differences and provide examples of how the experimental parameters of specific projects determine which method is most suitable.
Amino Acids↗
Engineering proteins for thermostability: the use of sequence alignments versus rational design and directed evolution.
With the advent of directed evolution techniques, protein engineering has received a fresh impetus. Engineering proteins for thermostability is a particularly exciting and challenging field, as it is crucial for broadening the industrial use of recombinant proteins. In addition to directed evolution, a variety of partially successful rational concepts for engineering thermostability have been developed in the past. Recent results suggest that amino acid sequence comparisons of mesophilic proteins alone can be used efficiently to engineer thermostable proteins. The potential benefits of the underlying, semirational 'consensus concept' are compared with those of rational design and directed evolution approaches.
Directed Molecular Evolution↗
Towards engineering proteins by site-directed incorporation in vivo of non-natural amino acids.
Altering protein structure via the techniques of protein engineering has already allowed the development of proteins displaying both modified and novel activities. The only limitation of conventional site-directed mutagenesis, the cornerstone of protein engineering, is that substitutions are restricted to the 20 naturally occurring, proteinogenic amino acids. However, the discovery of a 21st amino acid, selenocysteine, and the development of novel in vitro translation systems have demonstrated that considerably more substitutions are possible. To this end, a number of experimental approaches have been developed that allow the incorporation of synthetic amino acids into proteins. Some of these have already been successfully applied in vitro and efforts to transfer this technology to in vivo systems are now underway.
Amino Acids↗
Understanding cytochrome c function: engineering protein structure by semisynthesis.
Semisynthesis is a method of protein engineering that relies primarily on chemical manipulations of peptide fragments derived from natural proteins to produce intermediates for reassembly into defined analogs. In this respect it differs fundamentally from total chemical synthesis, but like it, it has unique capabilities for introducing noncoded amino acids or site-specific labels into the protein product. A favorite subject for semisynthesis is the redox protein cytochrome c: a vital link in the respiratory chain. The tactics that have evolved in the half dozen laboratories so engaged are briefly described. More than 100 semisynthetic analogs of this protein have resulted from this effort and have contributed to our understanding of the way in which the function of cytochrome c is determined by its three-dimensional structure. Questions concerning the folding and stability of the protein, the setting of heme redox potential, control and specificity of interaction with potential redox partners, and the mechanism of electron transfer are considered. A growing synergy between this approach and genetic methods will ensure that protein engineering by semisynthesis continues to increase our knowledge of this and other proteins.
Animals↗
Engineering proteins that bind, move, make and break DNA.
Recent protein engineering efforts have generated artificial transcription factors that bind new target DNA sequences and enzymes that modify DNA at new target sites. Zinc-finger-based transcription factors are favored targets for design; important technological advances in their construction and numerous biotechnological applications have been reported. Other notable advances include the generation of endonucleases and recombinases with altered specificities, made by innovative combinatorial and evolutionary protein engineering strategies. An unexpectedly high tolerance to mutation in the active sites of DNA polymerases is being exploited to engineer polymerases to incorporate artificial nucleotides or to display other, nonnatural activities.
DNA↗
Enamel structure properties controlled by engineered proteins in transgenic mice.
UNLABELLED: Amelogenin protein has regulatory effects on enamel biofabrication in mammalian tooth. Using teeth obtained from transgenic mice that express two separate protein-engineered versions of amelogenins, we made structure-nanomechanical properties correlations and showed 21% hardness and 24% elastic modulus degradation compared with the age-matched wildtype littermates. We attribute the inferior properties to the disorganization of the protein matrix resulting in defective mineral formation. INTRODUCTION: Enamel is a bioceramic initiated by the biosynthesis of a complex mixture of proteins that undergoes self-assembly to produce a super molecular ensemble that controls the nucleation and habit of the crystalline mineral phase. Ultimately, the inorganic crystals grow to almost fully replace the organic phase. This biofabrication process occurs at physiologic conditions of pH, temperature, pressure, and ion concentration and results in the hardest tissue in the vertebrate body, with the largest and longest substituted-hydroxyapatite crystals known to biomineralizing systems. The most abundant protein of forming mammalian enamel, amelogenin, has been shown to have a significant regulatory effect on this complex process. MATERIALS AND METHODS: In this work, we present the effect of protein engineering of amelogenin on the mechanical properties of the resultant mouse enamel. We have produced two types of transgenic animals that express separate versions of amelogenin proteins that lack the required self-assembly domains. The resultant matured enamel was extensively characterized for its mechanical properties at the nanoscale by means of nanoindentation and atomic force microscopy (AFM). These techniques have enabled us to probe the mechanical properties that are representative of a single enamel rod. RESULTS: Our nanoindentation measurements have revealed that the altered amelogenin with dysfunctional self-assembly properties resulted in a degradation by as much as 21% in hardness and 24% in elastic modulus compared with the age-matched wildtype littermates. Furthermore, the enamel formed by these defective proteins is found to display a decrease in indentation surface pile-up volume by up to 32%. CONCLUSIONS: We attribute these inferior mechanical properties for the enamel grown by the engineered amelogenins to result from the disorganization of the nanospheres formed in the protein matrix starting at the mineral nucleation stage with a consequential alteration to the fully grown mineral component. By engineering the properties of proteins that contribute to the nanoscale level of hierarchy in enamel biomineralization, it is possible to regulate the properties of the resulting bioceramic at the mesoscale level of the tissue.
Amelogenin↗
Protein engineering study of protein L by simulation.
We examine the ability of our recently introduced minimalist protein model to reproduce experimentally measured thermodynamic and kinetic changes upon sequence mutation in the well-studied immunoglobulin-binding protein L. We have examined five different sequence mutations of protein L that are meant to mimic the same mutation type studied experimentally: two different mutations which disrupt the natural preference in the beta-hairpin #1 and beta-hairpin #2 turn regions, two different helix mutants where a surface polar residue in the alpha-helix has been mutated to a hydrophobic residue, and a final mutant to further probe the role of nonnative hydrophobic interactions in the folding process. These simulated mutations are analyzed in terms of various kinetic and thermodynamic changes with respect to wild type, but in addition we evaluate the structure-activity relationship of our model protein based on the phi-value calculated from both the kinetic and thermodynamic perspectives. We find that the simulated thermodynamic phi-values reproduce the experimental trends in the mutations studied and allow us to circumvent the difficult interpretation of the complicated kinetics of our model. Furthermore, the level of resolution of the model allows us to directly predict what experiments seek in regard to protein engineering studies of protein folding--namely the residues or portions of the polypeptide chain that contribute to the crucial step in the folding of the wild-type protein.
Bacterial Proteins↗
Putting engineering back into protein engineering: bioinformatic approaches to catalyst design.
Complex multivariate engineering problems are commonplace and not unique to protein engineering. Mathematical and data-mining tools developed in other fields of engineering have now been applied to analyze sequence-activity relationships of peptides and proteins and to assist in the design of proteins and peptides with specified properties. Decreasing costs of DNA sequencing in conjunction with methods to quickly synthesize statistically representative sets of proteins allow modern heuristic statistics to be applied to protein engineering. This provides an alternative approach to expensive assays or unreliable high-throughput surrogate screens.
Algorithms↗
Potential use of additivity of mutational effects in simplifying protein engineering.
The problem of rationally engineering protein molecules can be simplified where effects of mutations on protein function are additive. Crystal structures of single and double mutants in the hydrophobic core of gene V protein indicate that structural and functional effects of core mutations are additive when the regions structurally influenced by the mutations do not substantially overlap. These regions of influence can provide a simple basis for identifying sets of mutations that will show additive effects.
Coliphages↗
Unnatural ligands for engineered proteins: new tools for chemical genetics.
Small molecules that modulate the activity of biological signaling molecules can be powerful probes of signal transduction pathways. Highly specific molecules with high affinity are difficult to identify because of the conserved nature of many protein active sites. A newly developed approach to discovery of such small molecules that relies on protein engineering and chemical synthesis has yielded powerful tools for the study of a wide variety of proteins involved in signal transduction (G-proteins, protein kinases, 7-transmembrane receptors, nuclear hormone receptors, and others). Such chemical genetic tools combine the advantages of traditional genetics and the unparalleled temporal control over protein function afforded by small molecule inhibitors/activators that act at diffusion controlled rates with targets.
Alleles↗