Genetic Encoding of Fluoro-l-tryptophans for Site-Specific Detection of Conformational Heterogeneity in Proteins by NMR Spectroscopy.

The substitution of a single hydrogen atom in a protein by fluorine yields a site-specific probe for sensitive detection by 19F nuclear magnetic resonance (NMR) spectroscopy, where the absence of background signal from the protein facilitates the detection of minor conformational species. We developed genetic encoding systems for the site-selective incorporation of 4-fluorotryptophan, 5-fluorotryptophan, 6-fluorotryptophan, and 7-fluorotryptophan in response to an amber stop codon and used them to investigate conformational heterogeneity in a designed amino acid binding protein and in flaviviral NS2B-NS3 proteases. These proteases have been shown to present variable conformations in X-ray crystal structures, including flips of the indole side chains of tryptophan residues. The 19F NMR spectra of different fluorotryptophan isomers installed at the conserved site of Trp83 indicate that the indole ring flip is common in flaviviral NS2B-NS3 proteases in the apo state and suppressed by an active-site inhibitor.

Genetic Encoding of 7-Aza-l-tryptophan: Isoelectronic Substitution of a Single CH-Group in a Protein for a Nitrogen Atom for Site-Selective Isotope Labeling.

Genetic encoding of a noncanonical amino acid (ncAA) in an in vivo expression system requires an aminoacyl-tRNA synthetase that specifically recognizes the ncAA, while the ncAA must not be recognized by the canonical protein expression machinery. We succeeded in genetically encoding 7-aza-tryptophan (7AW), which is isoelectronic with tryptophan. The system is fully orthogonal to protein expression in Escherichia coli, enabling high-yielding site-selective isotope labeling in vivo. 7AW is readily synthesized from serine and 7-aza-indole using a tryptophan synthetase ß-subunit (TrpB) mutant, affording easy access to isotope-labeled 7AW. Using labeled 7AW produced from 15N/13C-labeled serine, we produced 7AW mutants of the 25 kDa Zika virus NS2B-NS3 protease. 15N-HSQC spectra display single cross-peaks at chemical shifts near those observed for the wild-type protein labeled with 15N/13C-tryptophan, confirming the structural integrity of the protein and yielding straightforward NMR resonance assignments for site-specific probing.

Probing Ligand Binding Sites on Large Proteins by Nuclear Magnetic Resonance Spectroscopy of Genetically Encoded Non-Canonical Amino Acids.

N6-(((trimethylsilyl)-methoxy)carbonyl)-l-lysine (TMSK) and N6-trifluoroacetyl-l-lysine (TFAK) are non-canonical amino acids, which can be installed in proteins by genetic encoding. In addition, we describe a new aminoacyl-tRNA synthetase specific for N6-(((trimethylsilyl)methyl)-carbamoyl)-l-lysine (TMSNK), which is chemically more stable than TMSK. Using the dimeric SARS-CoV-2 main protease (Mpro) as a model system with three different ligands, we show that the 1H and 19F nuclei of the solvent-exposed trimethylsilyl and CF3 groups produce intense signals in the nuclear magnetic resonance (NMR) spectrum. Their response to active-site ligands differed significantly when positioned near rather than far from the active site. Conversely, the NMR probes failed to confirm the previously reported binding site of the ligand pelitinib, which was found to enhance the activity of Mpro by promoting the formation of the enzymatically active dimer. In summary, the amino acids TMSK, TMSNK, and TFAK open an attractive path for site-specific NMR analysis of ligand binding to large proteins of limited stability and at low concentrations.

Genetic Encoding of Cyanopyridylalanine for In-Cell Protein Macrocyclization by the Nitrile-Aminothiol Click Reaction.

Cyanopyridylalanines are non-canonical amino acids that react with aminothiol compounds under physiological conditions in a biocompatible manner without requiring added catalyst. Here we present newly developed aminoacyl-tRNA synthetases for genetic encoding of meta- and para-cyanopyridylalanine to enable the site-specific attachment of a wide range of different functionalities. The outstanding utility of the cyanopyridine moiety is demonstrated by examples of i) post-translational functionalization of proteins, ii) in-cell macrocyclization of peptides and proteins, and iii) protein stapling. The biocompatible nature of the protein ligation chemistry enabled by the cyanopyridylalanine amino acid opens a new path to specific in vivo protein modifications in complex biological environments.

Site-Specific Incorporation of 7-Fluoro-L-tryptophan into Proteins by Genetic Encoding to Monitor Ligand Binding by 19F NMR Spectroscopy.

A mutant aminoacyl-tRNA synthetase identified by a library selection system affords site-specific incorporation of 7-fluoro-L-tryptophan in response to an amber stop codon. The enzyme allows the production of proteins with a single hydrogen atom replaced by a fluorine atom as a sensitive nuclear magnetic resonance (NMR) probe. The substitution of a single hydrogen atom by another element that is as closely similar in size and hydrophobicity as possible minimizes possible perturbations in the structure, stability, and solubility of the protein. The fluorine atom enables site-selective monitoring of the protein response to ligand binding by 19F NMR spectroscopy, as demonstrated with the Zika virus NS2B-NS3 protease.

Through-Space Scalar 19F-19F Couplings between Fluorinated Noncanonical Amino Acids for the Detection of Specific Contacts in Proteins.

Fluorine atoms are known to display scalar 19F-19F couplings in nuclear magnetic resonance (NMR) spectra when they are sufficiently close in space for nonbonding orbitals to overlap. We show that fluorinated noncanonical amino acids positioned in the hydrophobic core or on the surface of a protein can be linked by scalar through-space 19F-19F (TSJFF) couplings even if the 19F spins are in the time average separated by more than the van der Waals distance. Using two different aromatic amino acids featuring CF3 groups, O-trifluoromethyl-tyrosine and 4-trifluoromethyl-phenylalanine, we show that 19F-19F TOCSY experiments are sufficiently sensitive to detect TSJFF couplings between 2.5 and 5 Hz in the 19 kDa protein PpiB measured on a two-channel 400 MHz NMR spectrometer with a regular room temperature probe. A quantitative J evolution experiment enables the measurement of TSJFF coupling constants that are up to five times smaller than the 19F NMR line width. In addition, a new aminoacyl-tRNA synthetase was identified for genetic encoding of N6-(trifluoroacetyl)-l-lysine (TFA-Lys) and 19F-19F TOCSY peaks were observed between two TFA-Lys residues incorporated into the proteins AncCDT-1 and mRFP despite high solvent exposure and flexibility of the TFA-Lys side chains. With the ready availability of systems for site-specific incorporation of fluorinated amino acids into proteins by genetic encoding, 19F-19F interactions offer a straightforward way to probe the spatial proximity of selected sites without any assignments of 1H NMR resonances.

Genetic Encoding of N6-(((Trimethylsilyl)methoxy)carbonyl)-l-lysine for NMR Studies of Protein-Protein and Protein-Ligand Interactions.

Trimethylsilyl (TMS) groups present outstanding NMR probes of biological macromolecules as they produce intense singlets in 1H NMR spectra near 0 ppm, where few other proton resonances occur. We report a system for genetic encoding of N6-(((trimethylsilyl)methoxy)carbonyl)-l-lysine (TMSK) for site-specific incorporation into proteins. The system is based on pyrrolysyl-tRNA synthetase mutants, which deliver proteins with high yield and purity in vivo and in cell-free protein synthesis. As the TMS signal can readily be identified in 1D 1H NMR spectra of high-molecular weight systems without the need of isotopic labeling, TMSK delivers an excellent site-specific NMR probe for the study of protein structure and function, which is both inexpensive and convenient. We demonstrate the utility of TMSK to detect ligand binding, measure the rate of conformational change, and assess protein dimerization by paramagnetic relaxation enhancement. In addition, we present a system for dual incorporation of two different unnatural amino acids (TMSK and O-tert-butyl-tyrosine) in the same protein in quantities sufficient for NMR spectroscopy. Close proximity of the TMS and tert-butyl groups was readily detected by nuclear Overhauser effects.

Genetic Encoding of para-Pentafluorosulfanyl Phenylalanine: A Highly Hydrophobic and Strongly Electronegative Group for Stable Protein Interactions.

SF5Phe, para-pentafluorosulfanyl phenylalanine, is an unnatural amino acid with extreme physicochemical properties, which is stable in physiological conditions. Here we present newly developed aminoacyl-tRNA synthetases that enable genetic encoding of SF5Phe for site-specific incorporation into proteins in high yields. Owing to the SF5 moiety's dichotomy of strong polarity and high hydrophobicity, the unnatural amino acid forms specific and strong interactions in proteins. The potential of SF5Phe in protein research is illustrated by (i) increasing the binding affinity of a consensus pentapeptide motif toward the ß subunit of Escherichia coli DNA polymerase III holoenzyme by mutation of a phenylalanine to a SF5Phe residue, (ii) site-specifically adhering ß-cyclodextrin to the surface of ubiquitin, and (iii) selective detection of 19F-19F nuclear Overhauser effects in the Escherichia coli peptidyl-prolyl cis/trans-isomerase B following mutation of two phenylalanine residues in the core of the protein to SF5Phe. With increasing use of the SF5 moiety in pharmaceutical chemistry, this general method of functionalizing proteins with SF5 groups opens unique opportunities for structural biology and in vivo studies.

Nucleoside-linked shunt products in the reaction catalyzed by the class C radical S-adenosylmethionine methyltransferase NosN.

NosN is a class C radical S-adenosylmethionine (SAM) methyltransferase (RSMT) involved in the biosynthesis of nosiheptide, a clinically interesting thiopeptide antibiotic produced by Streptomyces actuosus. NosN employs an unprecedented catalytic mechanism, in which SAM is converted to 5'-methylthioadenosine (MTA) as a direct methyl donor. In this study, we report identification of several nucleoside-linked shunt products in the NosN-catalyzed reaction. Comparative analysis of NosN and the class A RSMT RlmN and further density functional theory (DFT) calculations reveal important mechanistic insights into the catalyses of the two types of enzymes, showing that the radical intermediates generated by similar pathways can have very diverse reactivities. This investigation provides strong evidence supporting the previous mechanistic proposal of NosN catalysis, validating the presence of a key radical adduct that results from the addition of an MTA-derived methylene radical onto the C4 of the indolyl substrate.

The Catalytic Mechanism of the Class C Radical S-Adenosylmethionine Methyltransferase NosN.

S-Adenosylmethionine (SAM) is one of the most common co-substrates in enzyme-catalyzed methylation reactions. Most SAM-dependent reactions proceed through an SN 2 mechanism, whereas a subset of them involves radical intermediates for methylating non-nucleophilic substrates. Herein, we report the characterization and mechanistic investigation of NosN, a class C radical SAM methyltransferase involved in the biosynthesis of the thiopeptide antibiotic nosiheptide. We show that, in contrast to all known SAM-dependent methyltransferases, NosN does not produce S-adenosylhomocysteine (SAH) as a co-product. Instead, NosN converts SAM into 5'-methylthioadenosine as a direct methyl donor, employing a radical-based mechanism for methylation and releasing 5'-thioadenosine as a co-product. A series of biochemical and computational studies allowed us to propose a comprehensive mechanism for NosN catalysis, which represents a new paradigm for enzyme-catalyzed methylation reactions.

Reactivity of the nitrogen-centered tryptophanyl radical in the catalysis by the radical SAM enzyme NosL.

The radical SAM tryptophan (Trp) lyase NosL involved in nosiheptide biosynthesis catalyzes two parallel reactions, converting l-Trp to 3-methyl-2-indolic acid (MIA) and to dehydroglycine and 3-methylindole, respectively. The two parallel reactions diverge from a nitrogen-centered tryptophanyl radical intermediate. Here we report an investigation on the intrinsic reactivity of the tryptophanyl radical using a chemical model study and DFT calculations. The kinetics of the formation and fragmentation of this nitrogen-centered radical in NosL catalysis were also studied in detail. Our analysis explains the intriguing catalytic promiscuity of NosL and highlights the remarkable role this enzyme plays in achieving an energetically highly unfavorable transformation.

Emerging Diversity of the Cobalamin-Dependent Methyltransferases Involving Radical-Based Mechanisms.

Cobalamins comprise a group of cobalt-containing organometallic cofactors that play important roles in cellular metabolism. Although many cobalamin-dependent methyltransferases (e.g., methionine synthase MetH) have been extensively studied, a new group of methyltransferases that are cobalamin-dependent and utilize radical chemistry in catalysis is just beginning to be appreciated. In this Concept article, we summarize recent advances in the understanding of the radical-based and cobalamin-dependent methyltransferases and discuss the functional and mechanistic diversity of this emerging class of enzymes.