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.

Ligand-induced conformational changes in protein molecules detected by sum-frequency generation.

We present the first demonstration of ligand-induced conformational changes in a biological molecule, a protein, by sum-frequency generation (SFG). Constructs of KRas protein were prepared by selectively deuterating residues of a single amino acid type using isotope-labeled amino acids and cell-free protein synthesis. By attaching labeled protein to a supported bilayer membrane via a His-tag to Ni-NTA-bearing lipids, we ensured that single layers of ordered molecules were formed while preserving the protein's native structure.

A lanthanide tag for a complementary set of pseudocontact shifts.

Pseudocontact shifts (PCS) generated by paramagnetic lanthanide ions deliver powerful restraints for protein structure analysis by NMR spectroscopy. We present a new lanthanide tag that generates different PCSs than that of a related tag, which differs in structure by a single oxygen atom. It is highly reactive towards cysteine and performs well in turn-on luminescence and in EPR spectroscopy.

(2,4)-5-Fluoroleucine, (2,4)-5-Fluoroleucine, and 5,5'-Difluoroleucine in PpiB: Protein Production, F NMR, and Ligand Sensing Enhanced by the γ-Gauche Effect.

Global substitution of leucine for analogues containing CHF instead of methyl groups delivers proteins with multiple sites for monitoring by F nuclear magnetic resonance (NMR) spectroscopy. The 19 kDa peptidyl-prolyl isomerase B (PpiB) was prepared with uniform high-level substitution of leucine by (2,4)-5-fluoroleucine, (2,4)-5-fluoroleucine, or 5,5'-difluoroleucine. The stability of the samples toward thermal denaturation was little altered compared to the wild-type protein.

Conformational Preferences of the Non-Canonical Amino Acids (2,4)-5-Fluoroleucine, (2,4)-5-Fluoroleucine, and 5,5'-Difluoroleucine in a Protein.

Proteins produced with leucine analogues, where CHF groups substitute specific methyl groups, can readily be probed by F NMR spectroscopy. As CF and CH groups are similar in hydrophobicity and size, fluorinated leucines are expected to cause minimal structural perturbation, but the impact of fluorine on the rotational freedom of CHF groups is unclear. We present high-resolution crystal structures of peptidyl-prolyl - isomerase B (PpiB) prepared with uniform high-level substitution of leucine by (2,4)-5-fluoroleucine, (2,4)-5-fluoroleucine, or 5,5'-difluoroleucine.

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.

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 (F) as a nuclear spin label using high-field (W-band/3.4 T) ENDOR spectroscopy.

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 () 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.

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 Ca 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 Ca ions in the CaM mutant N60D by a Gd(III) ion.

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 C/N/D isotopologues requiring solely readily available and inexpensive isotope containing reagents such as NaBD, carbon-C dioxide and sodium azide-1-N. 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 , could be incorporated into proteins this way.

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.

NT*-HRV3CP: An optimized construct of human rhinovirus 14 3C protease for high-yield expression and fast affinity-tag cleavage.

The human rhinovirus 14 3C protease (HRV3C protease), in fusion with glutathione S-transferase also referred to as PreScission™ protease, is a cysteine protease of particular interest for affinity tag removal from fusion proteins due to its stringent recognition sequence specificity (LEVLFQ/GX) and superior activity at low temperature. Here we report the expression, purification and use of a fusion construct of HRV3C protease, NT*-HRV3CP, that affords high expression yield in E. coli (over 300 mg/L cell culture), facile single-step purification, high solubility (>10 mg/mL) and excellent storage properties.

Tracking Conformational Changes in Calmodulin in vitro, in Cell Extract, and in Cells by Electron Paramagnetic Resonance Distance Measurements.

It is an open question whether the conformations of proteins sampled in dilute solutions are the same as in the cellular environment. Here we address this question by double electron-electron resonance (DEER) distance measurements with Gd(III) spin labels to probe the conformations of calmodulin (CaM) in vitro, in cell extract, and in human HeLa cells. Using the CaM mutants N53C/T110C and T34C/T117C labeled with maleimide-DOTA-Gd(III) in the N- and C-terminal domains, we observed broad and varied interdomain distance distributions.

Small Gd(III) Tags for Gd(III)-Gd(III) Distance Measurements in Proteins by EPR Spectroscopy.

The C7-Gd and C8-Gd tags are compact hydrophilic cyclen-based lanthanide tags for conjugation to cysteine residues in proteins. The tags are enantiomers, which differ in the configuration of the 2-hydroxylpropyl pendant arms coordinating the lanthanide ion. Here, we report the electron paramagnetic resonance (EPR) performance of the C7-Gd ( S configuration) and C8-Gd ( R configuration) tags loaded with Gd(III) on two mutants of the homodimeric ERp29 protein.

Luminescent Alkyne-Bearing Terbium(III) Complexes and Their Application to Bioorthogonal Protein Labeling.

Two new bifunctional macrocyclic chelate ligands that form luminescent terbium(III) complexes featuring an alkyne group for conjugation to (bio)molecules via the Cu(I)-catalyzed "click" reaction were synthesized. Upon ligation, the complexes exhibit a significant luminescent enhancement when excited at the λ(max) of the "clicked" products. To demonstrate the utility of the complexes for luminescent labeling, they were conjugated in vitro to E.

A New Gd(3+) Spin Label for Gd(3+)-Gd(3+) Distance Measurements in Proteins Produces Narrow Distance Distributions.

Gd(3+) tags have been shown to be useful for performing distance measurements in biomolecules via the double electron-electron resonance (DEER) technique at Q- and W-band frequencies. We introduce a new cyclen-based Gd(3+) tag that exhibits a relatively narrow electron paramagnetic resonance (EPR) spectrum, affording high sensitivity, and which yields exceptionally narrow Gd(3+)-Gd(3+) distance distributions in doubly tagged proteins owing to a very short tether. Both the maxima and widths of distance distributions measured for tagged mutants of the proteins ERp29 and T4 lysozyme, featuring Gd(3+)-Gd(3+) distances of ca.

Pulse EPR-enabled interpretation of scarce pseudocontact shifts induced by lanthanide binding tags.

Pseudocontact shifts (PCS) induced by tags loaded with paramagnetic lanthanide ions provide powerful long-range structure information, provided the location of the metal ion relative to the target protein is known. Usually, the metal position is determined by fitting the magnetic susceptibility anisotropy (Δχ) tensor to the 3D structure of the protein in an 8-parameter fit, which requires a large set of PCSs to be reliable. In an alternative approach, we used multiple Gd(3+)-Gd(3+) distances measured by double electron-electron resonance (DEER) experiments to define the metal position, allowing Δχ-tensor determinations from more robust 5-parameter fits that can be performed with a relatively sparse set of PCSs.

Generation of pseudocontact shifts in proteins with lanthanides using small "clickable" nitrilotriacetic acid and iminodiacetic acid tags.

Pseudocontact shifts (PCS) induced by paramagnetic lanthanide ions provide unique long-range structural information in nuclear magnetic resonance (NMR) spectra, but the site-specific attachment of lanthanide tags to proteins remains a challenge. Here we incorporated p-azido-phenylalanine (AzF) site-specifically into the proteins ubiquitin and GB1, and ligated the AzF residue with alkyne derivatives of small nitrilotriacetic acid and iminodiacetic acid tags using the Cu(I) -catalysed "click" reaction. These tags form lanthanide complexes with no or only a small net charge and produced sizeable PCSs with paramagnetic lanthanide ions in all mutants tested.