Inter-residue through-space scalar F-F couplings between CHF groups in a protein.

Using cell-free protein synthesis, the protein G B1 domain (GB1) was prepared with uniform high-level substitution of leucine by (2 ,4 )-5-fluoroleucine (FLeu1), (2 ,4 )-5-fluoroleucine (FLeu2), or 5,5-difluoroleucine (diFLeu). nuclear magnetic resonance (NMR) spectra showed chemical shift ranges spanning more than 9 . Through-space scalar couplings between groups arising from transient fluorine-fluorine contacts are readily manifested in [ , ]-TOCSY spectra.

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.

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.

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

The metallo--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-C-indole and the protein with lanthanoid tags at three different sites. The magnetic susceptibility anisotropy () tensors were determined by measuring pseudocontact shifts (PCSs) of backbone amide protons.

A Chiral Lanthanide Tag for Stable and Rigid Attachment to Single Cysteine Residues in Proteins for NMR, EPR and Time-Resolved Luminescence Studies.

A lanthanide-binding tag site-specifically attached to a protein presents a tool to probe the protein by multiple spectroscopic techniques, including nuclear magnetic resonance, electron paramagnetic resonance and time-resolved luminescence spectroscopy. Here a new stable chiral Ln tag, referred to as C12, is presented for spontaneous and quantitative reaction with a cysteine residue to generate a stable thioether bond. The synthetic protocol of the tag is relatively straightforward, and the tag is stable for storage and shipping.

Synthesis of C/F/H labeled indoles for use as tryptophan precursors for protein NMR spectroscopy.

Synthesis of indoles labeled with 13C-1H and 13C-19F spin pairs is described. All syntheses utilize inexpensive carbon-13C dioxide as the 13C isotope source. Ruthenium-mediated ring-closing metathesis is the key step in construction of the 13C containing indole carbocycle.