Delineating the conformational landscape of the adenosine A2A receptor during G protein coupling.

G-protein-coupled receptors (GPCRs) represent a ubiquitous membrane protein family and are important drug targets. Their diverse signaling pathways are driven by complex pharmacology arising from a conformational ensemble rarely captured by structural methods. Here, fluorine nuclear magnetic resonance spectroscopy (19F NMR) is used to delineate key functional states of the adenosine A2A receptor (A2AR) complexed with heterotrimeric G protein (Gαsß1γ2) in a phospholipid membrane milieu. Analysis of A2AR spectra as a function of ligand, G protein, and nucleotide identifies an ensemble represented by inactive states, a G-protein-bound activation intermediate, and distinct nucleotide-free states associated with either partial- or full-agonist-driven activation. The Gßγ subunit is found to be critical in facilitating ligand-dependent allosteric transmission, as shown by 19F NMR, biochemical, and computational studies. The results provide a mechanistic basis for understanding basal signaling, efficacy, precoupling, and allostery in GPCRs.

Structure, dynamics, and redox reactivity of an all-purpose flavodoxin.

The flavodoxin of Rhodopseudomonas palustris CGA009 (Rp9Fld) supplies highly reducing equivalents to crucial enzymes such as hydrogenase, especially when the organism is iron-restricted. By acquiring those electrons from photodriven electron flow via the bifurcating electron transfer flavoprotein, Rp9Fld provides solar power to vital metabolic processes. To understand Rp9Fld's ability to work with diverse partners, we solved its crystal structure. We observed the canonical flavodoxin (Fld) fold and features common to other long-chain Flds but not all the surface loops thought to recognize partner proteins. Moreover, some of the loops display alternative structures and dynamics. To advance studies of protein-protein associations and conformational consequences, we assigned the 19F NMR signals of all five tyrosines (Tyrs). Our electrochemical measurements show that incorporation of 3-19F-Tyr in place of Tyr has only a modest effect on Rp9Fld's redox properties even though Tyrs flank the flavin on both sides. Meanwhile, the 19F probes demonstrate the expected paramagnetic effect, with signals from nearby Tyrs becoming broadened beyond detection when the flavin semiquinone is formed. However, the temperature dependencies of chemical shifts and linewidths reveal dynamics affecting loops close to the flavin and regions that bind to partners in a variety of systems. These coincide with patterns of amino acid type conservation but not retention of specific residues, arguing against detailed specificity with respect to partners. We propose that the loops surrounding the flavin adopt altered conformations upon binding to partners and may even participate actively in electron transfer.

Positive residues of the SARS-CoV-2 fusion domain are key contributors to the initiation of membrane fusion.

SARS-CoV-2 is one of the most infectious viruses ever recorded. Despite a plethora of research over the last several years, the viral life cycle is still not well understood, particularly membrane fusion. This process is initiated by the fusion domain (FD), a highly conserved stretch of amino acids consisting of a fusion peptide (FP) and fusion loop (FL), which in synergy perturbs the target cells' lipid membrane to lower the energetic cost necessary for fusion. In this study, through a mutagenesis-based approach, we have investigated the basic residues within the FD (K825, K835, R847, K854) utilizing an in vitro fusion assay and 19F NMR, validated by traditional 13C 15N techniques. Alanine and charge-conserving mutants revealed every basic residue plays a highly specific role within the mechanism of initiating fusion. Intriguingly, K825A led to increased fusogenecity which was found to be correlated to the number of amino acids within helix one, further implicating the role of this specific helix within the FD's fusion mechanism. This work has found basic residues to be important within the FDs fusion mechanism and highlights K825A, a specific mutation made within the FD of the SARS-CoV-2 spike protein, as requiring further investigation due to its potential to contribute to a more virulent strain of SARS-CoV-2.

Micromolar fluoride contamination arising from glass NMR tubes and a simple solution for biomolecular applications.

Fluorine (19F) NMR is emerging as an invaluable analytical technique in chemistry, biochemistry, structural biology, material science, drug discovery, and medicine, especially due to the inherent rarity of naturally occurring fluorine in biological, organic, and inorganic compounds. Here, we revisit the under-reported problem of fluoride leaching from new and unused glass NMR tubes. We characterised the leaching of free fluoride from various types of new and unused glass NMR tubes over the course of several hours and quantify this contaminant to be at micromolar concentrations for typical NMR sample volumes across multiple glass types and brands. We find that this artefact is undetectable for samples prepared in quartz NMR tubes within the timeframes of our experiments. We also observed that pre-soaking new glass NMR tubes combined with rinsing removes this contamination below micromolar levels. Given the increasing popularity of 19F NMR across a wide range of fields, increasing popularity of single-use screening tubes, the long collection times required for relaxation studies and samples of low concentrations, and the importance of avoiding contamination in all NMR experiments, we anticipate that our simple solution will be useful to biomolecular NMR spectroscopists.

Fluorine-19 labeling of the tryptophan residues in the G protein-coupled receptor NK1R using the 5-fluoroindole precursor in Pichia pastoris expression.

In NMR spectroscopy of biomolecular systems, the use of fluorine-19 probes benefits from a clean background and high sensitivity. Therefore, 19F-labeling procedures are of wide-spread interest. Here, we use 5-fluoroindole as a precursor for cost-effective residue-specific introduction of 5-fluorotryptophan (5F-Trp) into G protein-coupled receptors (GPCRs) expressed in Pichia pastoris. The method was successfully implemented with the neurokinin 1 receptor (NK1R). The 19F-NMR spectra of 5F-Trp-labeled NK1R showed one well-separated high field-shifted resonance, which was assigned by mutational studies to the "toggle switch tryptophan". Residue-selective labeling thus enables site-specific investigations of this functionally important residue. The method described here is inexpensive, requires minimal genetic manipulation and can be expected to be applicable for yeast expression of GPCRs at large.

Fluorinated Pharmaceutical and Pesticide Photolysis: Investigating Reactivity and Identifying Fluorinated Products by Combining Computational Chemistry,19F NMR, and Mass Spectrometry.

Fluorinated breakdown products from photolysis of pharmaceuticals and pesticides are of environmental concern due to their potential persistence and toxicity. While mass spectrometry workflows have been shown to be useful in identifying products, they fall short for fluorinated products and may miss up to 90% of products. Studies have shown that 19F NMR measurements assist in identifying and quantifying reaction products, but this protocol can be further developed by incorporating computations. Density functional theory was used to compute 19F NMR shifts for parent and product structures in photolysis reactions. Computations predicted NMR spectra of compounds with an R2 of 0.98. Computed shifts for several isolated product structures from LC-HRMS matched the experimental shifts with <0.7 ppm error. Multiple products including products that share the same shift that were not previously reported were identified and quantified using computational shifts, including aliphatic products in the range of -80 to -88 ppm. Thus, photolysis of fluorinated pharmaceuticals and pesticides can result in compounds that are polyfluorinated alkyl substances (PFAS), including aliphatic-CF3 or vinyl-CF2 products derived from heteroaromatic-CF3 groups. C-F bond-breaking enthalpies and electron densities around the fluorine motifs agreed well with the experimentally observed defluorination of CF3 groups. Combining experimental-computational 19F NMR allows quantification of products identified via LC-HRMS without the need for authentic standards. These results have applications for studies of environmental fate and analysis of fluorinated pharmaceuticals and pesticides in development.

19F-NMR Probing of Ion-Induced Conformational Changes in Detergent-Solubilized and Nanodisc-Reconstituted NCX_Mj.

Consecutive interactions of 3Na+ or 1Ca2+ with the Na+/Ca2+ exchanger (NCX) result in an alternative exposure (access) of the cytosolic and extracellular vestibules to opposite sides of the membrane, where ion-induced transitions between the outward-facing (OF) and inward-facing (IF) conformational states drive a transport cycle. Here, we investigate sub-state populations of apo and ion-bound species in the OF and IF states by analyzing detergent-solubilized and nanodisc-reconstituted preparations of NCX_Mj with 19F-NMR. The 19F probe was covalently attached to the cysteine residues at entry locations of the cytosolic and extracellular vestibules. Multiple sub-states of apo and ion-bound species were observed in nanodisc-reconstituted (but not in detergent-solubilized) NCX_Mj, meaning that the lipid-membrane environment preconditions multiple sub-state populations toward the OF/IF swapping. Most importantly, ion-induced sub-state redistributions occur within each major (OF or IF) state, where sub-state interconversions may precondition the OF/IF swapping. In contrast with large changes in population redistributions, the sum of sub-state populations within each inherent state (OF or IF) remains nearly unchanged upon ion addition. The present findings allow the further elucidation of structure-dynamic modules underlying ion-induced conformational changes that determine a functional asymmetry of ion access/translocation at opposite sides of the membrane and ion transport rates concurring physiological demands.

NMR Investigation of Protein-Carbohydrate Interactions: The Recognition of Glycans by Galectins Engineered with Fluorotryptophan Residues.

Fluorine (19 F) incorporation into glycan-binding proteins (lectins) has been achieved and exploited to monitor the binding to carbohydrate ligands by nuclear magnetic resonance (NMR) spectroscopy. Galectins are a family of lectins that bind carbohydrates, generally with weak affinities, through a combination of intermolecular interactions including a key CH-π stacking involving a conserved tryptophan residue. Herein, Galectin-3 (Gal3) and Galectin-8 (Gal8) with one and two carbohydrate recognition domains (CRDs), respectively, were selected. Gal3 contains one Trp, whereas Gal8 contains three, one at each binding site and a third one not involved in sugar binding; these were substituted by the corresponding F-Trp analogues. The presence of fluorine did not significantly modify the affinity for glycan binding, which was in slow exchange on the 19 F NMR chemical-shift timescale, even for weak ligands, and allowed binding events taking place at two different binding sites within the same lectin to be individualized.

Probing Peptidylprolyl Bond cis/trans Status Using Distal 19 F NMR Reporters.

A method for measuring peptidylprolyl bond cis-trans conformational status in peptide models is described, using 4-fluorophenylalanine (4FPhe) as a distal reporter for 19 F NMR. The %cis-Pro population was measured for peptides of the general structure Ac-X-Pro-Z-Ala-Ala-4FPhe (X and Z are proteinogenic amino acids) at pH 7.4, and provided conformational populations consistent with literature values obtained by more complex methods. This approach was applied to probe the prolyl bond status in pentapeptide models of the intrinsically disordered C-terminal region of α-synuclein, which mirrored the preferences in the Ac-X-Pro-Z-Ala-4FPhe models. Advantageously, the 19 F reporter group does not need to be adjacent to or attached to proline to provide quantifiable signals and distal 4-fluorophenylalanines can be placed so as not to influence prolyl bond conformation. Finally, we demonstrated that the prolyl bond status is not significantly affected by pH when there are ionisable amino acid residues at the carboxyl side of proline, which makes 19 F NMR an invaluable tool with which to study proline isomerism at a range of pHs and in different solvents and buffers.

19 F-Labeled Probes for Recognition-Enabled Chromatographic 19 F NMR.

The NMR technique is among the most powerful analytical methods for molecular structural elucidation, process monitoring, and mechanistic investigations; however, the direct analysis of complex real-world samples is often hampered by crowded NMR spectra that are difficult to interpret. The combination of fluorine chemistry and supramolecular interactions leads to a unique detection method named recognition-enabled chromatographic (REC) 19 F NMR, where interactions between analytes and 19 F-labeled probes are transduced into chromatogram-like 19 F NMR signals of discrete chemical shifts. In this account, we summarize our endeavor to develop novel 19 F-labeled probes tailored for separation-free multicomponent analysis. The strategies to achieve chiral discrimination, sensitivity enhancement, and automated analyte identification will be covered. The account will also provide a detailed discussion of the underlying principles for the design of molecular probes for REC 19 F NMR where appropriate.

QM assisted ML for 19F NMR chemical shift prediction.

BACKGROUND: Ligand-observed 19F NMR detection is an efficient method for screening libraries of fluorinated molecules in fragment-based drug design campaigns. Screening fluorinated molecules in large mixtures makes 19F NMR a high-throughput method. Typically, these mixtures are generated from pools of well-characterized fragments. By predicting 19F NMR chemical shift, mixtures could be generated for arbitrary fluorinated molecules facilitating for example focused screens. METHODS: In a previous publication, we introduced a method to predict 19F NMR chemical shift using rooted fluorine fingerprints and machine learning (ML) methods. Having observed that the quality of the prediction depends on similarity to the training set, we here propose to assist the prediction with quantum mechanics (QM) based methods in cases where compounds are not well covered by a training set. RESULTS: Beyond similarity, the performance of ML methods could be associated with individual features in compounds. A combination of both could be used as a procedure to split input data sets into those that could be predicted by ML and those that required QM processing. We could show on a proprietary fluorinated fragment library, known as LEF (Local Environment of Fluorine), and a public Enamine data set of 19F NMR chemical shifts that ML and QM methods could synergize to outperform either method individually. Models built on Enamine data, as well as model building and QM workflow tools, can be found at https://github.com/PatrickPenner/lefshift and https://github.com/PatrickPenner/lefqm .

Wavelength-Dependent UV-LED Photolysis of Fluorinated Pesticides and Pharmaceuticals.

The wavelength dependence of photoproduct formation and quantum yields was evaluated for fluorinated pesticides and pharmaceuticals using UV-light emitting diodes (LEDs) with 255, 275, 308, 365, and 405 nm peak wavelengths. The fluorinated compounds chosen were saflufenacil, penoxsulam, sulfoxaflor, fluoxetine, 4-nitro-3-trifluoromethylphenol (TFM), florasulam, voriconazole, and favipiravir, covering key fluorine motifs (benzylic-CF3, heteroaromatic-CF3, aryl-F, and heteroaromatic-F). Quantum yields for the compounds were consistently higher for UV-C as compared to UV-A wavelengths and did not show the same trend as molar absorptivity. For all compounds except favipiravir and TFM, the fastest degradation was observed using 255 or 275 nm light, despite the low power of the LEDs. Using quantitative 19F NMR, fluoride, trifluoroacetate, and additional fluorinated byproducts were tracked and quantified. Trifluoroacetate was observed for both Ar-CF3 and Het-CF3 motifs and increased at longer wavelengths for Het-CF3. Fluoride formation from Het-CF3 was significantly lower as compared to other motifs. Ar-F and Het-F motifs readily formed fluoride at all wavelengths. For Het-CF3 and some Ar-CF3 motifs, 365 nm light produced either a greater number of or different major products. Aliphatic-CF2/CF3 products were stable under all wavelengths. These results assist in selecting the most efficient wavelengths for UV-LED degradation and informing future design of fluorinated compounds.

Species-differences in the in vitro biotransformation of trifluoroethene (HFO-1123).

1,1,2-Trifluoroethene (HFO-1123) is anticipated for use as a refrigerant with low global warming potential. Inhalation studies on HFO-1123 in rats indicated a low potential for toxicity (NOAELs ≥ 20,000 ppm). In contrast, single inhalation exposure of Goettingen® minipigs (≥ 500 ppm) and New Zealand white rabbits (≥ 1250 ppm) resulted in severe toxicity. It has been suggested that these pronounced species-differences in toxicity may be attributable to species-differences in biotransformation of HFO-1123 via the mercapturic acid pathway. Therefore, the overall objective of this study was to evaluate species-differences in glutathione (GSH) dependent in vitro metabolism of HFO-1123 in susceptible versus less susceptible species and humans as a basis for human risk assessment. Biotransformation of HFO-1123 to S-(1,1,2-trifluoroethyl)-L-glutathione (1123-GSH) and subsequent cysteine S-conjugate ß-lyase-mediated cleavage of the corresponding cysteine conjugate (1123-CYS) was monitored in hepatic and renal subcellular fractions of mice, rats, minipigs, rabbits, and humans. While 1123-GSH formation occurred at higher rates in rat and rabbit liver S9 compared to minipig and human S9, increased ß-lyase cleavage of 1123-CYS was observed in minipig kidney cytosol as compared to cytosolic fractions of other species. Increased ß-lyase activity in minipig cytosol was accompanied by time-dependent formation of monofluoroacetic acid (MFA), a highly toxic compound that interferes with cellular energy production via inhibition of aconitase. Consistent with the significantly lower ß-lyase activity in human cytosols, the intensity of the MFA signal in human cytosols was only a fraction of the signal obtained in minipig subcellular fractions. Even though the inconsistencies between GSH and ß-lyase-dependent metabolism do not allow to draw a firm conclusion on the overall contribution of the mercapturic acid pathway to HFO-1123 biotransformation and toxicity in vivo, the ß-lyase data suggest that humans may be less susceptible to HFO-1123 toxicity compared to minipigs.

Computational 19 F NMR of trifluoromethyl derivatives of alkenes, pyrimidines, and indenes.

The 19 F NMR chemical shifts of 13 trifluoromethyl derivatives of alkenes, pyrimidines, and indenes were calculated at the DFT level using the BhandHLYP, BHandH, PBE, PBE0, O3LYP, B3LYP, KT2, and KT3 functionals in combination with the pcS-2 basis set. Best result was documented for the BHandHLYP functional: The mean absolute error (MAE) of 0.66 ppm for the scaled values was achieved for the range of about 20 ppm. Solvent, vibrational, and relativistic corrections were found to be rather small, especially when taken in combination, generally demonstrating a slight decrease in the difference between calculated and experimental fluorine chemical shifts. As a measure of the practical importance of these compounds, one should recall that the growing number of life science products that contain trifluoromethyl groups provides a continuing driving force for the development of an effective methodology that enables both regio- and stereoselective introduction of trifluoromethyl groups into both aliphatic and aromatic systems.

Diffusion 19F-NMR of Nanofluorides: In Situ Quantification of Colloidal Diameters and Protein Corona Formation in Solution.

The NMR-detectability of elements of organic ligands that stabilize colloidal inorganic nanocrystals (NCs) allow the study of their diffusion characteristics in solutions. Nevertheless, these measurements are sensitive to dynamic ligand exchange and often lead to overestimation of diffusion coefficients of dispersed colloids. Here, we present an approach for the quantitative assessment of the diffusion properties of colloidal NCs based on the NMR signals of the elements of their inorganic cores. Benefiting from the robust 19F-NMR signals of the fluorides in the core of colloidal CaF2 and SrF2, we show the immunity of 19F-diffusion NMR to dynamic ligand exchange and, thus, the ability to quantify, with high accuracy, the colloidal diameters of different types of nanofluorides in situ. With the demonstrated ability to characterize the formation of protein corona at the surface of nanofluorides, we envision that this study can be extended to additional formulations and applications.

Fluorinated Human Serum Albumin as Potential 19F Magnetic Resonance Imaging Probe.

Fluorinated human serum albumin conjugates were prepared and tested as potential metal-free probes for 19F magnetic resonance imaging (MRI). Each protein molecule was modified by several fluorine-containing compounds via the N-substituted natural acylating reagent homocysteine thiolactone. Albumin conjugates retain the protein's physical and biological properties, such as its 3D dimensional structure, aggregation ability, good solubility, proteolysis efficiency, biocompatibility, and low cytotoxicity. A dual-labeled with cyanine 7 fluorescence dye and fluorine reporter group albumin were synthesized for simultaneous fluorescence imaging and 19F MRI. The preliminary in vitro studies show the prospects of albumin carriers for multimodal imaging.

Multi-Site Conformational Exchange in the Synthetic Neomycin-Sensing Riboswitch Studied by 19 F NMR.

The synthetic neomycin-sensing riboswitch interacts with its cognate ligand neomycin as well as with the related antibiotics ribostamycin and paromomycin. Binding of these aminoglycosides induces a very similar ground state structure in the RNA, however, only neomycin can efficiently repress translation initiation. The molecular origin of these differences has been traced back to differences in the dynamics of the ligand:riboswitch complexes. Here, we combine five complementary fluorine based NMR methods to accurately quantify seconds to microseconds dynamics in the three riboswitch complexes. Our data reveal complex exchange processes with up to four structurally different states. We interpret our findings in a model that shows an interplay between different chemical groups in the antibiotics and specific bases in the riboswitch. More generally, our data underscore the potential of 19 F NMR methods to characterize complex exchange processes with multiple excited states.

Computational protocol for predicting 19 F NMR chemical shifts for PFAS and connection to PFAS structure.

Per- and polyfluoroalkyl substances (PFAS) are robust "forever" chemicals that have become global environmental contaminants due to their inability to degrade using traditional techniques. In addition to the persistent nature of PFAS, the structural and functional diversity in PFAS creates a unique challenge in identification and remediation. Their identification is further complicated by the absence of standards for many PFAS. This work is aimed at developing a protocol for computing and establishing accurate 19 F NMR chemical shifts for PFAS using density functional theory (DFT), which can aid in the identification of PFAS. The impact of solvation and basis sets was evaluated by comparing the computed data with the experimental measurements. Results showed the addition of dispersion corrections in the methodology improve the accuracy of calculated NMR parameters within 4 ppm of the experimental values. Adding a second diffuse function and additional polarization did not improve the accuracy, likely because of the electronegativity of fluorine which does not allow the electron density of fluorine atoms to be polarized. The inclusion of various implicit solvation (DMSO, chloroform, and water) yielded negligible differences in accuracy, and were overall less accurate than the gas phase calculations. The most accurate methodology was then applied to more environmentally relevant PFAS, and the impact of helical nature on the NMR signatures was evaluated. The implication of this work is to be able to improve the identification of structurally diverse PFAS using the 19 F NMR.

An inexpensive density functional theory-based protocol to predict accurate 19 F-NMR chemical shifts.

Thanks to its advantages, 19 F-NMR is an increasingly popular technique for the structural characterization of F-containing molecules, among which polymers, materials, fluorophores, pharmaceuticals, and so forth. However, the computational calculation of the 19 F-NMR chemical shifts, both for prediction and interpretation of experimental spectra, remains a challenge. In this work a density functional theory (DFT) based protocol for the calculation of the chemical shifts is established within the framework of the gauge-independent atomic orbital method, upon verifying the performance of Hartree-Fock and 60 DFT functionals coupled with seven different basis sets. The benchmark is conducted using two sets of molecules, namely one used for testing methods and another used for probing; the former set consists of 134 molecules, the latter 50, yet both of them with F in different chemical environments. Following Bally-Rablen-Tantillo strategy, the scaling parameters and other statistical quantities were computed for each method upon least squares linear regression between experimental and computed chemical shifts. The designed computational workflow is computationally inexpensive and represents a significant improvement with respect to the current state of the art.

Overexpression of recombinant proteins containing non-canonical amino acids in Vibrio natriegens: p-azido-L-phenylalanine as coupling site for 19F-tags.

Vibrio natriegens is the fastest growing organism identified so far. The minimum doubling time of only 9.4 min, the ability to utilize over 60 different carbon sources and its non-pathogenic properties make it an interesting alternative to E. coli as a new production host for recombinant proteins. We investigated the ability of the engineered V. natriegens strain, Vmax™ Express, to incorporate the non-canonical amino acid (ncAA) p-azido-L-phenylalanine (AzF) into recombinant proteins for NMR applications. AzF was incorporated into enhanced yellow fluorescent protein (EYFP) and MlaC, an intermembrane transport protein, by stop codon suppression. AzF incorporation into EYFP resulted in an improved suppression efficiency (SE) of up to 35.5 ± 0.8% and a protein titer of 26.7 ± 0.7 mg/L. The expression levels of MlaC-AzF even exceeded those of E. coli BL21 cells. For the recording of 1H-15N and 19F NMR spectra, EYFP-AzF was expressed and isotopically labeled in minimal medium and the newly introduced azido-group was used as coupling site for NMR sensitive 19F-tags. Our findings show that Vmax is a flexible expression host, suitable for the incorporation of ncAAs in recombinant proteins with the potential to surpass protein yields of E. coli. The presented method suggests the implementation of V. natriegens for expression of isotopically labeled proteins containing ncAAs, which can be chemically modified for the application in protein-observed 19F-NMR.