The goal of xenobiology is to design biological systems endowed with unusual biochemical functions, whereas enzymology concerns the study of enzymes, the workhorses of biocatalysis. Biocatalysis employs enzymes and organisms to perform useful biotransformations in synthetic chemistry and biotechnology. During the past few years, the effects of incorporating noncanonical amino acids (ncAAs) into enzymes with potential applications in biocatalysis have been increasingly investigated. In this Review, we provide an overview of the effects of new chemical functionalities that have been introduced into proteins to improve various facets of enzymatic catalysis. We also discuss future research avenues that will complement unnatural mutagenesis with standard protein engineering to produce novel and versatile biocatalysts with applications in synthetic organic chemistry and biotechnology.

Deciphering the fluorine code is how we describe not only the focus of this Account, but also the systematic approach to studying the impact of fluorine's incorporation on the properties of peptides and proteins used by our groups and others. The introduction of fluorine has been shown to impart favorable, but seldom predictable, properties to peptides and proteins, but up until about two decades ago the outcomes of fluorine modification of peptides and proteins were largely left to chance. Driven by the motivation to extend the application of the unique properties of the element fluorine from medicinal and agro chemistry to peptide and protein engineering we have established extensive research programs that enable the systematic investigation of effects that accompany the introduction of fluorine into this class of biopolymers. The introduction of fluorine into amino acids offers a universe of options for modifications with regard to number and position of fluorine substituents in the amino acid side chain. Moreover, it is important to emphasize that the consequences of incorporating the C-F bond into a biopolymer can be attributed to two distinct yet related phenomena: (i) the fluorine substituent can directly engage in intermolecular interactions with its environment and/or (ii) the other functional groups present in the molecule can be influenced by the electron withdrawing nature of this element (intramolecular) and in turn interact differently with their immediate environment (intermolecular). Based on our studies, we have shown that a change in number and/or position of as subtle as one single fluorine substituent has the power to considerably modify key properties of amino acids such as hydrophobicity, polarity, and secondary structure propensity. These properties are crucial factors in peptide and protein engineering, and thus, fluorinated amino acids can be applied to fine-tune properties such as protein folding, proteolytic stability, and protein-protein interactions provided we understand and become able to predict the outcome of a fluorine substitution in this context. With this Account, we attempt to analyze information we gained from our recent projects on how the nature of the fluorine atom and C-F bond influence four key properties of peptides and proteins: peptide folding, protein-protein interactions, ribosomal translation, and protease stability. These results impressively show why the introduction of fluorine creates a new class of amino acids with a repertoire of functionalities that is unique to the world of proteins and in some cases orthogonal to the set of canonical and natural amino acids. Our concluding statements aim to offer a few conserved design principles that have emerged from systematic studies over the last two decades; in this way, we hope to advance the field of peptide and protein engineering based on the judicious introduction of fluorinated building blocks.

Electrostatic interactions are essential for controlling the protein structure and function. Whereas so far experimental and theoretical efforts focused on the effect of local electrostatics, this work aims at elucidating the long-range modulation of electric fields in proteins upon binding to charged surfaces. The study is based on cytochrome c (Cytc) variants carrying nitrile reporters for the vibrational Stark effect that are incorporated into the protein via genetic engineering and chemical modification. The Cytc variants were thoroughly characterized with respect to possible structural perturbations due to labeling. For the proteins in solution, the relative hydrogen bond occupancy and the calculated electric fields, both obtained from molecular dynamics (MD) simulations, and the experimental nitrile stretching frequencies were used to develop a relationship for separating hydrogen-bonding and non-hydrogen-bonding electric field effects. This relationship provides an excellent description for the stable Cytc variants in solution. For the proteins bound to Au electrodes coated with charged self-assembled monolayers (SAMs), the underlying MD simulations can only account for the electric field changes Δ E due to the formation of the electrostatic SAM-Cytc complexes but not for the additional contribution, Δ E, representing the consequences of the potential drops over the electrode/SAM/protein interfaces. Both Δ E and Δ E, determined at distances between 20 and 30 Å with respect to the SAM surface, are comparable in magnitude to the non-hydrogen-bonding electric field in the unbound protein. This long-range modulation of the internal electric field may be of functional relevance for proteins in complexes with partner proteins (Δ E) and attached to membranes (Δ E + Δ E).

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Fluorine being not substantially present in the chemistry of living beings is an attractive element in tailoring novel chemical, biophysical, and pharmacokinetic properties of peptides and proteins. The hallmark of ribosome-mediated artificial amino acid incorporation into peptides and proteins is a broad substrate tolerance, which is assumed to rely on the absence of evolutionary pressure for efficient editing of artificial amino acids. We used the well-characterized editing proficient isoleucyl-tRNA synthetase (IleRS) from Escherichia coli to investigate the crosstalk of aminoacylation and editing activities against fluorinated amino acids. We show that translation of trifluoroethylglycine (TfeGly) into proteins is prevented by hydrolysis of TfeGly-tRNAIle in the IleRS post-transfer editing domain. The remarkable observation is that dissociation of TfeGly-tRNAIle from IleRS is significantly slowed down. This finding is in sharp contrast to natural editing reactions by tRNA synthetases wherein fast editing rates for the noncognate substrates are essential to outcompete fast aa-tRNA dissociation rates. Using a post-transfer editing deficient mutant of IleRS (IleRSAla10), we were able to achieve ribosomal incorporation of TfeGly in vivo. Our work expands the knowledge of ribosome-mediated artificial amino acid translation with detailed analysis of natural editing function against an artificial amino acid providing an impulse for further systematic investigations and engineering of the translation and editing of unusual amino acids.