Peter Kast

Protein design by directed evolution.

While nature evolved polypeptides over billions of years, protein design by evolutionary mimicry is progressing at a far more rapid pace. The mutation, selection, and amplification steps of the evolutionary cycle may be imitated in the laboratory using existing proteins, or molecules created de novo from random sequence space, as starting templates. However, the astronomically large number of possible polypeptide sequences remains an obstacle to identifying and isolating functionally interesting variants. Intelligently designed libraries and improved search techniques are consequently important for future advances. In this regard, combining experimental and computational methods holds particular promise for the creation of tailored protein receptors and catalysts for tasks unimagined by nature.

Investigating and Engineering Enzymes by Genetic Selection

Natural enzymes have arisen over millions of years by the gradual process of Darwinian evolution. The fundamental steps of evolution-mutation, selection, and amplification-can also be exploited in the laboratory to create and characterize protein catalysts on a human timescale. In vivo genetic selection strategies enable the exhaustive analysis of protein libraries with 10(10) different members, and even larger ensembles can be studied with in vitro methods. Evolutionary approaches can consequently yield statistically meaningful insight into the complex and often subtle interactions that influence protein folding, structure, and catalytic mechanism. Such methods are also being used increasingly as an adjunct to design, thus providing access to novel proteins with tailored catalytic activities and selectivities.

Evolution of a designed retro-aldolase leads to complete active site remodeling

Evolutionary advances are often fueled by unanticipated innovation. Directed evolution of a computationally designed enzyme suggests that dramatic molecular changes can also drive the optimization of primitive protein active sites. The specific activity of an artificial retro-aldolase was boosted >4,400 fold by random mutagenesis and screening, affording catalytic efficiencies approaching those of natural enzymes. However, structural and mechanistic studies reveal that the engineered catalytic apparatus, consisting of a reactive lysine and an ordered water molecule, was unexpectedly abandoned in favor of a new lysine residue in a substrate binding pocket created during the optimization process. Structures of the initial in silico design, a mechanistically promiscuous intermediate, and one of the most evolved variants highlight the importance of loop mobility and supporting functional groups in the emergence of the new catalytic center. Such internal competition between alternative reactive sites may have characterized the early evolution of many natural enzymes.

Efficient introduction of aryl bromide functionality into proteins in vivo

Artificial proteins can be engineered to exhibit interesting solid state, liquid crystal or interfacial properties and may ultimately serve as important alternatives to conventional polymeric materials. The utility of protein‐based materials is limited, however, by the availability of just the 20 amino acids that are normally recognized and utilized by biological systems; many desirable functional groups cannot be incorporated directly into proteins by biosynthetic means. In this study, we incorporate para‐bromophenylalanine (p‐Br‐phe) into a model target protein, mouse dihydrofolate reductase (DHFR), by using a bacterial phenylalanyl‐tRNA synthetase (PheRS) variant with relaxed substrate specificity. Coexpression of the mutant PheRS and DHFR in a phenylalanine auxotrophic Escherichia coli host strain grown in p‐Br‐phe‐supplemented minimal medium resulted in 88% replacement of phenylalanine residues by p‐Br‐phe; variation in the relative amounts of phe and p‐Br‐phe in the medium allows control of the degree of substitution by the analog. Protein expression yields of 20–25 mg/l were obtained from cultures supplemented with p‐Br‐phe; this corresponds to about two‐thirds of the expression levels characteristic of cultures supplemented with phe. The aryl bromide function is stable under the conditions used to purify DHFR and creates new opportunities for post‐translational derivatization of brominated proteins via metal‐catalyzed coupling reactions. In addition, bromination may be useful in X‐ray studies of proteins via the multiwavelength anomalous diffraction (MAD) technique.

Probing the Proteolytic Stability of β‐Peptides Containing α‐Fluoro‐ and α‐Hydroxy‐β‐Amino Acids

One of the benefits of β‐peptides as potential candidates for biological applications is their stability against common peptidases. Attempts have been made to rationalize this stability by altering the electron availability of a given amide carbonyl bond through the introduction of polar substituents at the α‐position of a single β‐amino acid. Such β‐amino acids (β‐homoglycine, β‐homoalanine), containing one or two fluorine atoms or a hydroxy group in the α‐position, were prepared in enantiopure form. A versatile method for preparing these α‐fluoro‐β‐amino acids by the homologation of appropriate α‐amino acids and C‐OH→C‐F or CO→CF2 substitution with DAST, is described. Consequently, a series of β‐peptides possessing an electronically modified residue at the N terminus or embedded within the chain was synthesized, and their proteolytic stability was investigated against a selection of enzymes. All ten β‐peptides tested were resilient to proteolysis. Introducing a polar, sterically undemanding group, into the α‐position of β‐amino acids in a β‐peptide chain does not appear to facilitate localized or general enzymatic degradation.

Robust design and optimization of retroaldol enzymes

Enzyme catalysts of a retroaldol reaction have been generated by computational design using a motif that combines a lysine in a nonpolar environment with water‐mediated stabilization of the carbinolamine hydroxyl and β‐hydroxyl groups. Here, we show that the design process is robust and repeatable, with 33 new active designs constructed on 13 different protein scaffold backbones. The initial activities are not high but are increased through site‐directed mutagenesis and laboratory evolution. Mutational data highlight areas for improvement in design. Different designed catalysts give different borohydride‐reduced reaction intermediates, suggesting a distribution of properties of the designed enzymes that may be further explored and exploited.

Is chorismate mutase a prototypic entropy trap? - Activation parameters for the Bacillus subtilis enzyme

Chorismate mutase is thought to accelerate the chorismate-to-prephenate rearrangement in part by significantly lowering the entropy barrier for the reaction. We have determined the activation parameters for the well-characterized Bacillus subtilis chorismate mutase and find that ΔS† (−9.1 ± 1.2 eu) is nearly as unfavorable as the activation entropy for the uncatalyzed process. Our results suggest that chorismate mutase catalysts show greater mechanistic versatility than commonly believed.

A simple selection strategy for evolving highly efficient enzymes

Combining tunable transcription with an enzyme-degradation tag affords an effective means to reduce intracellular enzyme concentrations from high to very low levels. Such fine-tuned control allows selection pressure to be systematically increased in directed-evolution experiments. This facilitates identification of mutants with wild-type activity, as shown here for an engineered chorismate mutase. Numerous selection formats and cell-based screening methodologies may benefit from the large dynamic range afforded by this easily implemented strategy.

Mechanistic Insights into the Isochorismate Pyruvate Lyase Activity of the Catalytically Promiscuous PchB from Combinatorial Mutagenesis and Selection

PchB from Pseudomonas aeruginosa possesses isochorismate pyruvate lyase (IPL) and weak chorismate mutase (CM) activity. Homology modeling based on a structurally characterized CM, coupled with randomization of presumed key active site residues (Arg54, Glu90, Gln91) and in vivo selection for CM activity, was used to derive mechanistic insights into the IPL activity of PchB. Mutation of Arg54 was incompatible with viability, and the CM and IPL activities of an engineered R54K variant were reduced 1,000-fold each. The observation that position 90 was tolerant to substitution but position 91 was essentially confined to Gln or Glu in functional variants rules out involvement of Glu90 in general base catalysis. Counter to the generally accepted mechanistic hypothesis for pyruvate lyases, we propose for PchB a rare [1,5]-sigmatropic reaction mechanism that invokes electrostatic catalysis in analogy to the [3,3]-pericyclic rearrangement of chorismate in CMs. A common catalytic principle for both PchB functions is also supported by the covariance of the catalytic parameters for the CM and IPL activities and the shared functional requirement for a protonated Glu91 in Q91E variants. The experiments demonstrate that focusing directed evolution strategies on the readily accessible surrogate activity of an enzyme can provide valuable insights into the mechanism of the primary reaction.

Searching sequence space for protein catalysts

Genetic selection was used to explore the probability of finding enzymes in protein sequence space. Large degenerate libraries were prepared by replacing all secondary structure units in a dimeric, helical bundle chorismate mutase with simple binary-patterned modules based on a limited set of four polar and four nonpolar residues. Two-stage in vivo selection yielded catalytically active variants possessing biophysical and kinetic properties typical of the natural enzyme even though ≈80% of the protein originates from the simplified modules and >90% of the protein consists of only eight different amino acids. This study provides a quantitative assessment of the number of sequences compatible with a given fold and implicates previously unidentified residues needed to form a functional active site. Given the extremely low incidence of enzymes in completely unbiased libraries, strategies that combine chemical information with genetic selection, like the one used here, may be generally useful in designing novel protein scaffolds with tailored activities.