Research

¹⁹F–Gd³⁺ ENDOR is used to map the full conformational distribution of Trp42 in dihedral space in a de novo luminescent lanthanide-binding protein. By combining genetically encoded fluorination with regularisation modelling, we extract ¹⁹F–Gd³⁺ distance distributions, revealing a minor rotamer population proximal to the lanthanide, key for photoactivity.

Noncanonical amino acids (ncAA) are promising as light-harvesting antennae for lanthanide luminescence in lanthanide-binding peptides and proteins. Here, we present empirical insights into antenna–lanthanide interactions, which reveal design principles of bright luminescent proteins. Peptides designed to act as lanthanide-binding tags (LBT) show a trade-off between sensitization and lanthanide-binding affinity. We generated a new protein, termed RF2, through computational design with nanomolar binding affinity and more than a 2-fold increase in terbium(III) luminescence relative to LBT. In this scaffold, 6-azatryptophan (6AW) achieved a 10-fold enhancement of the europium(III) luminescence in vivo. The RF2 6AW mutant also sensitizes the luminescence of dysprosium(III) and samarium(III). These results demonstrate the capability of de novo protein design to produce highly luminescent lanthanide-binding mini-proteins with a genetically encoded ncAA antenna.

Luminescence spectroscopies are attractive due to their sensitivity and selectivity. Polarised light provides added dimensions to luminescence data, leading to techniques that provide information about molecular structure and interactions. In this review the principles of steady-state fluorescence techniques, including fluorescence-detected circular dichroism, fluorescence-detected linear dichroism, fluorescence polarisation anisotropy, circularly polarised luminescence, and linearly polarised luminescence, are outlined and illustrated with examples of how they have been used to study biomolecules and their interactions with other molecules.

Aromatic ring flips in proteins provide a direct probe of local structural fluctuations, yet their rates are typically too fast for quantitative measurement by NMR spectroscopy. Here we show that site-specific incorporation of 2,3,4,5,6-pentafluoro-l-phenylalanine (F⁵Phe) reshapes the torsional energy landscape of aromatic side chains, slowing ring flips by over 2 orders of magnitude and shifting them into the slow-exchange regime accessible by ¹⁹F NMR. F⁵Phe can be genetically encoded with high fidelity and minimal structural perturbation, as confirmed by high-resolution X-ray crystallography across multiple proteins. The resulting ¹⁹F NMR spectra enable direct, quantitative measurements of ring-flip kinetics without the need for isotope labeling or complex multidimensional experiments. Application to a diverse set of proteins demonstrates that ring-flip rates vary widely even within the same hydrophobic cluster, revealing highly localized conformational fluctuations rather than global unfolding events. Pressure-dependent measurements yield small activation volumes, indicating that the structural rearrangements enabling ring flips are spatially confined. Ligand binding and protein–protein interactions modulate ring-flip rates in a site-specific manner, providing a sensitive readout of allosteric effects on local protein malleability. These results establish fluorinated aromatic amino acids as a general chemical strategy to engineer dynamic observables in proteins, transforming aromatic ring flips into a broadly applicable probe of local conformational dynamics and allostery.

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. The absorption maximum of 4-CN-Trp is distinct from Trp, allowing the selective excitation of its intense fluorescence. 4-CN-Trp fluoresces in the visible range with an intensity rivalling that of 7-hydroxy-coumarin. Crystal structures of maltose binding protein demonstrate near-complete structural conservation when a native buried Trp residue is replaced by 4-CN-Trp. Besides presenting an inconspicuous tag for live cell microscopy, the intense fluorescence of 4-CN-Trp enables measurements of subnanomolar ligand binding affinities in isotropic solution, as demonstrated by the complex between rapamycin and the peptidyl–prolyl isomerase FKBP12 furnished with a 4-CN-Trp residue in the substrate binding pocket. Furthermore, 4-CN-Trp residues positioned at different locations of a protein containing multiple tryptophan residues permits using fluorescence quenching experiments to detect the proximity of individual Trp residues to the binding site of aromatic ligands.

It has recently been shown that distances > 20 Å can be measured between Gd³⁺ spin tags and ¹⁹F labels in proteins using ¹⁹F-ENDOR at 94 GHz. Here we examine the precision with which this method can locate the positions of (aromatic) amino acid side chains and, with the help of established simulation tools, determine the conformational space sampled by solvent-exposed chains in solution. First, using a novel set of fluorinated phenylalanine amino acids incorporated into the metalloprotein Calbindin D9k binding Gd³⁺, we show that triangulation of the corresponding ¹⁹F-ENDOR determined distances can precisely identify the conformation of buried side chains. The obtained conformation agrees with the same (single) orientation seen in the crystal structure of the native protein. In a second set of proteins labeled with a Gd³⁺ spin tag and noncanonical ¹⁹F-labeled amino acids, the splittings, lineshapes, and integrated intensities of the ¹⁹F-ENDOR signals were used to constrain the conformational space of the side chains initially identified by comprehensive rotamer simulations. This work provides the first example where ¹⁹F-ENDOR constraints have been used to accurately pinpoint side chain conformations in a protein with the help of differently fluorinated amino acids.

Protein phosphorylation is one of the most important posttranslational modifications altering the structure, stability, and activity of more than 13 000 human proteins. In this work, the phosphotyrosine mimetic pentafluorophosphato-difluoromethyl-phenylalanine (PF₅CF₂Phe) was genetically encoded and incorporated into three different proteins. Screening two libraries of orthogonal aminoacyl-tRNA synthetases identified enzymes enabling the efficient and specific incorporation of PF₅CF₂Phe into red fluorescent protein (RFP) via amber stop codon suppression. Two model proteins, human ubiquitin (Ubq) and the B1 immunoglobulin-binding domain of streptococcal protein G (GB1), were prepared with PF₅CF₂Phe mutations and investigated for potential interaction partners. While native GB1 showed no binding to protein tyrosine phosphatases (PTP), PF₅-GB1, with PF₅CF₂Phe at position 17, was a strong inhibitor of the phosphatases PTP1B and SHP2. PF₅-Ubq was produced and converted into the first example of a protein carrying the most prominent phosphotyrosine mimetic, phosphono-difluoromethyl phenylalanine (PO₃CF₂Phe). With increasing need in the biosciences to delineate the functions of complex phosphorylation patterns, genetic encoding of PF₅CF₂Phe yielding phosphoprotein mimetics opens unique opportunities for precise functional studies where site-specific and homogeneous protein modifications are required.

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.