Environmentally Ultrasensitive Fluorine Probe to Resolve Protein Conformational Ensembles by ¹⁹F NMR and Cryo-EM

Abstract

Limited chemical shift dispersion represents a significant barrier to studying multistate equilibria of large membrane proteins by 19F NMR. We describe a novel monofluoroethyl 19F probe that dramatically increases the chemical shift dispersion. The improved conformational sensitivity and line shape enable the detection of previously unresolved states in one-dimensional (1D) 19F NMR spectra of a 134 kDa membrane transporter. Changes in the populations of these states in response to ligand binding, mutations, and temperature correlate with population changes of distinct conformations in structural ensembles determined by single-particle cryo-electron microscopy (cryo-EM). Thus, 19F NMR can guide sample preparation to discover and visualize novel conformational states and facilitate image analysis and three-dimensional (3D) classification.

Modifying cellular properties via rational chemical design for unnatural oxygen reduction electrocatalysis of a cell

“…NMR and XPS measurements indicate that the atomic interaction site for Fe–TA complexes on Cyt c proteins is the S atom in the methionine 80 (Met 80) residue, supporting the theoretical calculation results. 32,33 The 400 MHz 1 H NMR spectra results show that the characteristic peaks at −2.3, −6.4, and −24.3 ppm assigned to the β-CH 2 and ε-CH 3 protons (Fig. S24†) of the axial Met 80 ligand undergo paramagnetic shifted resonances to high field (−2.4, −6.6, and −24.5 ppm) in Cyt c-Fe–TA 34–36 (Fig.…”

Unnatural Amino Acids for Biological Spectroscopy and Microscopy

“…Compared to other isotopes (i.e., 13 C, 15 N, and 31 P), the detection of 19 F-containing UAAs is less affected by the background due to almost 100% natural abundance of 19 F and its absence in organisms. Therefore, a large variety of 19 F-containing UAAs (Figure ) have been developed and used in NMR studies of proteins under both in vitro and in vivo conditions. , …”

Enlarging the scenario of site directed 19F labeling for NMR spectroscopy of biomolecules

“…3-bromo-1,1,1-trifluoroacetone (BTFA) 28 – 30 and analogues, 2,2,2-trifluoroethanethiol (TFET) 22 , 23 , 31 , 32 or 4-(perfluoro-tert-butyl)phenyliodoacetamide (PFP) 33 are among the most used molecules. Other recent and interesting developed fluorine tags were exploited to observe globular proteins directly inside human cells 34 and to increase the 19 F chemical shift dispersion 35 . However, cysteine modification can have some drawbacks, including the fact that in a number of proteins cysteines are in the active site and/or coordinate metal cofactors.…”