Measuring Nanometer Distances in Proteins and Rigid Rulers between F and Gd by Integration of F-ENDOR Signal Intensities.

F ENDOR is emerging as a powerful tool in structural biology for measuring distances in proteins labeled with F and a paramagnetic tag. Due to spin-spin relaxation and line width limitations, it has been difficult to determine intertag distances larger than about 15 Å. Using a set of geometrically well-defined rulers and spin-labeled proteins, we show that F-Gd distances up to 20 Å can be accessed by integrating the intensity of the ENDOR spectrum, with distances approaching 30 Å potentially in reach as well.

Genetic Encoding of Fluorinated Analogues of Phenylalanine for F NMR Spectroscopy: Detection of Conformational Heterogeneity in Flaviviral NS2B-NS3 Proteases.

Substituting a single hydrogen atom in a protein by fluorine provides a probe for site-specific sensing by F nuclear magnetic resonance (NMR) spectroscopy with minimal impact on the properties of the protein. Genetic encoding systems are presented for five different fluorinated analogues of phenylalanine: 2-, 3-, 4-fluorophenylalanine, 2,6-difluorophenylalanine, and 3,5-difluorophenylalanine. The systems allow the installation of each of these amino acids with high fidelity during in vivo bacterial protein synthesis in response to an amber stop codon.

Rendering Proteins Fluorescent Inconspicuously: Genetically Encoded 4-Cyanotryptophan Conserves Their Structure and Enables the Detection of Ligand Binding Sites.

Cyanotryptophans (CN-Trp) are privileged multimodal reporters on protein structure. They are similar in size to the canonical amino acid tryptophan and some of them exhibit bright fluorescence which responds sensitively to changes in the environment. We selected aminoacyl-tRNA synthetases specific for 4-, 5-, 6-, and 7-CN-Trp for high-yield in vivo production of proteins with a single, site-specifically introduced nitrile label.

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 F 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.

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 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 , enabling high-yielding site-selective isotope labeling .

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

-(((trimethylsilyl)-methoxy)carbonyl)-l-lysine (TMSK) and -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 -(((trimethylsilyl)methyl)-carbamoyl)-l-lysine (TMSNK), which is chemically more stable than TMSK. Using the dimeric SARS-CoV-2 main protease (M) as a model system with three different ligands, we show that the H and F nuclei of the solvent-exposed trimethylsilyl and CF groups produce intense signals in the nuclear magnetic resonance (NMR) spectrum.

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.

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

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

Fluorine atoms are known to display scalar F-F 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 F-F () couplings even if the F spins are in the time average separated by more than the van der Waals distance. Using two different aromatic amino acids featuring CF groups, -trifluoromethyl-tyrosine and 4-trifluoromethyl-phenylalanine, we show that F-F TOCSY experiments are sufficiently sensitive to detect couplings between 2.

Genetic Encoding of -(((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 H NMR spectra near 0 ppm, where few other proton resonances occur. We report a system for genetic encoding of -(((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 and in cell-free protein synthesis.

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

SFPhe, -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 SFPhe for site-specific incorporation into proteins in high yields. Owing to the SF moiety's dichotomy of strong polarity and high hydrophobicity, the unnatural amino acid forms specific and strong interactions in proteins.

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.

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 S 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.

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.