Electrostatic Contribution to 19F Chemical Shifts in Fluorotryptophans in Proteins.

DFT calculations indicate that the 19F chemical shifts of aromatic rings containing single fluorine substituents are sensitive to the electric fields and electric field gradients at the position of the fluorine atom. The present work explores whether long-range structure restraints can be gained from changes in 19F chemical shifts following mutations of charged to uncharged residues. 19F chemical shifts of fluorotryptophan residues were measured in two different proteins, GB1 and the NT* domain, following mutations of single asparagine residues to aspartic acid. Four different versions of fluorotryptophan were investigated, including 4-, 5-, 6-, and 7-fluorotryptophan, which were simultaneously installed by cell-free protein synthesis using 4-, 5-, 6-, and 7-fluoroindole as precursors for the tryptophan synthase present in the S30 extract. For comparison, the 1H chemical shifts of the corresponding nonfluorinated protein mutants produced with 13C-labeled tryptophan were also measured. The results show that the 19F chemical shifts respond more sensitively to the charge mutations than the 1H chemical shifts in the nonfluorinated references, but the chemical shift changes were much smaller than predicted by DFT calculations of fluoroindoles in the electric field of a partial charge in vacuum, indicating comprehensive dielectric shielding by water and protein. No straightforward correlation with the location of the charge mutation could be established.

Cell-free synthesis of proteins with selectively 13C-labelled methyl groups from inexpensive precursors.

The novel eCell system maintains the activity of the entire repertoire of metabolic Escherichia coli enzymes in cell-free protein synthesis. We show that this can be harnessed to produce proteins with selectively 13C-labelled amino acids from inexpensive 13C-labelled precursors. The system is demonstrated with selective 13C labelling of methyl groups in the proteins ubiquitin and peptidyl-prolyl cis-trans isomerase B. Starting from 3-13C-pyruvate, 13C-HSQC cross-peaks are obtained devoid of one-bond 13C-13C scalar couplings. Starting from 2-13C-methyl-acetolactate, single methyl groups of valine and leucine are labelled. Labelling efficiencies are 70 % or higher, and the method allows us to produce perdeuterated proteins with protonated methyl groups in a residue-selective manner. The system uses the isotope-labelled precursors sparingly and is readily scalable.

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.

Improved spectral resolution of [13C,1H]-HSQC spectra of aromatic amino acid residues in proteins produced by cell-free synthesis from inexpensive 13C-labelled precursors.

Cell-free protein synthesis using eCells allows production of amino acids from inexpensive 13C-labelled precursors. We show that the metabolic pathway converting pyruvate, glucose and erythrose into aromatic amino acids is maintained in eCells. Judicious choice of 13C-labelled starting material leads to proteins, where the sidechains of aromatic amino acids display [13C,1H]-HSQC cross-peaks free of one-bond 13C-13C couplings. Selective 13C-labelling of tyrosine and phenylalanine residues is achieved simply by using different compositions of the reaction buffers.

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.

Short-range ENDOR distance measurements between Gd(III) and trifluoromethyl labels in proteins.

The measurement of distances in proteins can be challenging in the 5-20 Å range, which is outside those accessible through conventional NMR and EPR methods. Recently it was demonstrated that distances in this range could be measured between a nitroxide as a paramagnetic spin label and a nearby fluorine atom (19F) as a nuclear spin label using high-field (W-band/3.4 T) ENDOR spectroscopy. Here we show that such measurements can also be performed using a gadolinium ion (Gd3+) as the paramagnetic tag. Gd3+ has two advantages. (i) A greater electronic spin (S = 7/2) and fast electronic spin-lattice (T1) relaxation, improving sensitivity by allowing data to be collected at lower temperatures. (ii) A narrow EPR signal for the -½ ↔ ½ transition, and therefore no orientation selection artefacts. Signal intensities can be further enhanced by using a trifluoromethyl (C19F3) group instead of a single 19F atom. Using the protein calbindin D9k with a Ca2+ ion replaced by a Gd3+ ion and a trifluoromethylphenylalanine in position 50, we show that distances up to about 10 Å can be readily measured. Longer distances proved more difficult to measure due to variable electronic TM relaxation rates, which lead to broader Lorentzian ENDOR lineshapes. Gd3+ complexes (Gd3+ tags), which reliably display longer TM times, allow longer distances to be measured (8-16 Å). We also provide preliminary evidence that the intensity of ENDOR signals follows the predicted 1/r6 dependence, indicating that distances r > 20 Å can be measured by this method.

Antiviral cyclic peptides targeting the main protease of SARS-CoV-2.

Antivirals that specifically target SARS-CoV-2 are needed to control the COVID-19 pandemic. The main protease (Mpro) is essential for SARS-CoV-2 replication and is an attractive target for antiviral development. Here we report the use of the Random nonstandard Peptide Integrated Discovery (RaPID) mRNA display on a chemically cross-linked SARS-CoV-2 Mpro dimer, which yielded several high-affinity thioether-linked cyclic peptide inhibitors of the protease. Structural analysis of Mpro complexed with a selenoether analogue of the highest-affinity peptide revealed key binding interactions, including glutamine and leucine residues in sites S1 and S2, respectively, and a binding epitope straddling both protein chains in the physiological dimer. Several of these Mpro peptide inhibitors possessed antiviral activity against SARS-CoV-2 in vitro with EC50 values in the low micromolar range. These cyclic peptides serve as a foundation for the development of much needed antivirals that specifically target SARS-CoV-2.

DEER experiments reveal fundamental differences between calmodulin complexes with IQ and MARCKS peptides in solution.

Calmodulin (CaM) is a calcium-binding protein that regulates the function of many proteins by indirectly conferring Ca2+ sensitivity, and it undergoes a large conformational change on partners' binding. We compared the solution binding mode of the target peptides MARCKS and IQ by double electron-electron resonance (DEER) distance measurements and paramagnetic NMR. We combined nitroxide and Gd(III) spin labels, including specific substitution of one of the Ca2+ ions in the CaM mutant N60D by a Gd(III) ion. The binding of MARCKS to holo-CaM resulted neither in a closed conformation nor in a unique relative orientation between the two CaM domains, in contrast with the crystal structure. Binding of IQ to holo-CaM did generate a closed conformation. Using elastic network modeling and 12 distance restraints obtained from multiple holo-CaM/IQ DEER data, we derived a model of the solution structure, which is in reasonable agreement with the crystal structure.

Synthesis of fluorinated leucines, valines and alanines for use in protein NMR.

Efficient syntheses of fluorinated leucines, valines and alanines are described. The synthetic routes provide expedient access to various 13C/15N/D isotopologues requiring solely readily available and inexpensive isotope containing reagents such as NaBD4, carbon-13C dioxide and sodium azide-1-15N. The lightly fluorinated leucines and valines were found to be good substrates for cell-free protein expression and even 3-fluoroalanine, which is highly toxic to bacteria in vivo, could be incorporated into proteins this way. 19F-NMR spectra of the protein GB1 produced with these amino acids showed large chemical shift dispersions. Particularly high incorporation yields and clean 19F-NMR spectra were obtained for GB1 produced with valine residues, which had been synthesized with a single fluorine substituting a hydrogen stereospecifically in one of the methyl groups.

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.

Main protease mutants of SARS-CoV-2 variants remain susceptible to nirmatrelvir.

The COVID-19 pandemic continues to be a public health threat. Multiple mutations in the spike protein of emerging variants of SARS-CoV-2 appear to impact on the effectiveness of available vaccines. Specific antiviral agents are keenly anticipated but their efficacy may also be compromised in emerging variants. One of the most attractive coronaviral drug targets is the main protease (Mpro). A promising Mpro inhibitor of clinical relevance is the peptidomimetic nirmatrelvir (PF-07321332). We expressed Mpro of six SARS-CoV-2 lineages (C.37 Lambda, B.1.1.318, B.1.2, B.1.351 Beta, B.1.1.529 Omicron, P.2 Zeta), each of which carries a strongly prevalent missense mutation (G15S, T21I, L89F, K90R, P132H, L205V). Enzyme kinetics reveal that these Mpro variants are catalytically competent to a similar degree as the wildtype. We show that nirmatrelvir has similar potency against the variants as the wildtype. Our in vitro data suggest that the efficacy of the specific Mpro inhibitor nirmatrelvir is not compromised in current COVID-19 variants.

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.

Paramagnetic Chemical Probes for Studying Biological Macromolecules.

Paramagnetic chemical probes have been used in electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spectroscopy for more than four decades. Recent years witnessed a great increase in the variety of probes for the study of biological macromolecules (proteins, nucleic acids, and oligosaccharides). This Review aims to provide a comprehensive overview of the existing paramagnetic chemical probes, including chemical synthetic approaches, functional properties, and selected applications. Recent developments have seen, in particular, a rapid expansion of the range of lanthanoid probes with anisotropic magnetic susceptibilities for the generation of structural restraints based on residual dipolar couplings and pseudocontact shifts in solution and solid state NMR spectroscopy, mostly for protein studies. Also many new isotropic paramagnetic probes, suitable for NMR measurements of paramagnetic relaxation enhancements, as well as EPR spectroscopic studies (in particular double resonance techniques) have been developed and employed to investigate biological macromolecules. Notwithstanding the large number of reported probes, only few have found broad application and further development of probes for dedicated applications is foreseen.

Site-selective generation of lanthanoid binding sites on proteins using 4-fluoro-2,6-dicyanopyridine.

The paramagnetism of a lanthanoid tag site-specifically installed on a protein provides a rich source of structural information accessible by nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy. Here we report a lanthanoid tag for selective reaction with cysteine or selenocysteine with formation of a (seleno)thioether bond and a short tether between the lanthanoid ion and the protein backbone. The tag is assembled on the protein in three steps, comprising (i) reaction with 4-fluoro-2,6-dicyanopyridine (FDCP); (ii) reaction of the cyano groups with α-cysteine, penicillamine or ß-cysteine to complete the lanthanoid chelating moiety; and (iii) titration with a lanthanoid ion. FDCP reacts much faster with selenocysteine than cysteine, opening a route for selective tagging in the presence of solvent-exposed cysteine residues. Loaded with Tb3+ and Tm3+ ions, pseudocontact shifts were observed in protein NMR spectra, confirming that the tag delivers good immobilisation of the lanthanoid ion relative to the protein, which was also manifested in residual dipolar couplings. Completion of the tag with different 1,2-aminothiol compounds resulted in different magnetic susceptibility tensors. In addition, the tag proved suitable for measuring distance distributions in double electron-electron resonance experiments after titration with Gd3+ ions.

Localising individual atoms of tryptophan side chains in the metallo-ß-lactamase IMP-1 by pseudocontact shifts from paramagnetic lanthanoid tags at multiple sites.

The metallo-ß-lactamase IMP-1 features a flexible loop near the active site that assumes different conformations in single crystal structures, which may assist in substrate binding and enzymatic activity. To probe the position of this loop, we labelled the tryptophan residues of IMP-1 with 7-13C-indole and the protein with lanthanoid tags at three different sites. The magnetic susceptibility anisotropy (Δχ) tensors were determined by measuring pseudocontact shifts (PCSs) of backbone amide protons. The Δχ tensors were subsequently used to identify the atomic coordinates of the tryptophan side chains in the protein. The PCSs were sufficient to determine the location of Trp28, which is in the active site loop targeted by our experiments, with high accuracy. Its average atomic coordinates showed barely significant changes in response to the inhibitor captopril. It was found that localisation spaces could be defined with better accuracy by including only the PCSs of a single paramagnetic lanthanoid ion for each tag and tagging site. The effect was attributed to the shallow angle with which PCS isosurfaces tend to intersect if generated by tags and tagging sites that are identical except for the paramagnetic lanthanoid ion.

Localising nuclear spins by pseudocontact shifts from a single tagging site.

Ligating a protein at a specific site with a tag molecule containing a paramagnetic metal ion provides a versatile way of generating pseudocontact shifts (PCSs) in nuclear magnetic resonance (NMR) spectra. PCSs can be observed for nuclear spins far from the tagging site, and PCSs generated from multiple tagging sites have been shown to enable highly accurate structure determinations at specific sites of interest, even when using flexible tags, provided the fitted effective magnetic susceptibility anisotropy (Δχ) tensors accurately back-calculate the experimental PCSs measured in the immediate vicinity of the site of interest. The present work investigates the situation where only the local structure of a protein region or bound ligand is to be determined rather than the structure of the entire molecular system. In this case, the need for gathering structural information from tags deployed at multiple sites may be queried. Our study presents a computational simulation of the structural information available from samples produced with single tags attached at up to six different sites, up to six different tags attached to a single site, and in-between scenarios. The results indicate that the number of tags is more important than the number of tagging sites. This has important practical implications, as it is much easier to identify a single site that is suitable for tagging than multiple ones. In an initial experimental demonstration with the ubiquitin mutant S57C, PCSs generated with four different tags at a single site are shown to accurately pinpoint the location of amide protons in different segments of the protein.

Organoarsenic probes to study proteins by NMR spectroscopy.

Arsenical probes enable structural studies of proteins. We report the first organoarsenic probes for nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR) spectroscopy to study proteins in solutions. These probes can be attached to irregular loop regions. A lanthanide-binding tag induces sizable pseudocontact shifts in protein NMR spectra of a magnitude never observed for small paramagnetic probes before.

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.

Challenges of short substrate analogues as SARS-CoV-2 main protease inhibitors.

Specific anti-coronaviral drugs complementing available vaccines are urgently needed to fight the COVID-19 pandemic. Given its high conservation across the betacoronavirus genus and dissimilarity to human proteases, the SARS-CoV-2 main protease (Mpro) is an attractive drug target. SARS-CoV-2 Mpro inhibitors have been developed at unprecedented speed, most of them being substrate-derived peptidomimetics with cysteine-modifying warheads. In this study, Mpro has proven resistant towards the identification of high-affinity short substrate-derived peptides and peptidomimetics without warheads. 20 cyclic and linear substrate analogues bearing natural and unnatural residues, which were predicted by computational modelling to bind with high affinity and designed to establish structure-activity relationships, displayed no inhibitory activity at concentrations as high as 100 µM. Only a long linear peptide covering residues P6 to P5' displayed moderate inhibition (Ki = 57 µM). Our detailed findings will inform current and future drug discovery campaigns targeting Mpro.