Fast Magic-Angle-Spinning ¹⁹F Spin Exchange NMR for Determining Nanometer ¹⁹F–¹⁹F Distances in Proteins and Pharmaceutical Compounds

Abstract

Internuclear distances measured using NMR provide crucial constraints of three-dimensional structures but are often restricted to about 5 Å due to the weakness of nuclear-spin dipolar couplings. For studying macromolecular assemblies in biology and materials science, distance constraints beyond 1 nm will be extremely valuable. Here we present an extensive and quantitative analysis of the feasibility of F spin exchange NMR for precise and robust measurements of interatomic distances up to 1.6 nm at a magnetic field of 14.1 T, under 20-40 kHz magic-angle spinning (MAS). The measured distances are comparable to those achievable from paramagnetic relaxation enhancement but have higher precision, which is better than ±1 Å for short distances and ±2 Å for long distances. ForF spins with the same isotropic chemical shift but different anisotropic chemical shifts, intermediate MAS frequencies of 15-25 kHz without H irradiation accelerate spin exchange. For spectrally resolvedF-F spin exchange, H-F dipolar recoupling significantly speeds up F-F spin exchange. On the basis of data from five fluorinated synthetic, pharmaceutical, and biological compounds, we obtained two general curves for spin exchange between CF groups and between CF and CF groups. These curves allow F-F distances to be extracted from the measured spin exchange rates after taking into account F chemical shifts. These results demonstrate the robustness ofF spin exchange NMR for distance measurements in a wide range of biological and chemical systems.

Fast Magic‐Angle Spinning 19F NMR Spectroscopy of HIV‐1 Capsid Protein Assemblies

“…CSA parameters are also important for inter-fluorine distance measurements by spin exchange experiments. [14] Furthermore, our long-term goal is to combine experiment and quantum chemical calculations of 19 F CSA parameters in conjunction with 19 F- 19 F distance restraints for structure elucidation of large protein assemblies. While we have recently demonstrated the proof of principle on model crystalline tryptophans, [27] to apply this approach broadly it is necessary to establish a database of experimental 19 F CSA parameters for a range of proteins and protein assemblies.…”

“…For fluorine distance measurements by MAS NMR, several methods are available: RFDR [33] , CODEX, [40] and, more recently, PDSD [32] and CORD [41] . In a recent study at 14.1 T which employed PDSD and CORD mixing at a MAS frequency of 25 kHz, correlations were observed for distances as long as 16 Å for long CORD mixing times of 306 ms. [14] …”

“…The strong 19 F- 19 F dipolar couplings make fluorine well suited as a long-range distance probe in the solid state and distances up to 20 Å have been detected. [13–14] Fluorine is absent from any naturally occurring biological molecule, yet it can be readily and selectively incorporated into proteins, [15–17] largely without causing major structural perturbations. [18] 19 F NMR, both solution and solid-state, has therefore emerged as an essential method with broad applications in pharmaceutical chemistry (as ~30% of all drugs at present in the clinic contain fluorine), [19] chemical biology, [20] biochemistry, [16, 21–24] and materials science.…”

“…Only three recent studies used faster spinning frequencies. In two, resolution enhancements were observed at 35 and 40 kHz, [14, 26] while a report from our group demonstrated that frequencies of 40–60 kHz yielded significant line narrowing for fluorosubstituted tryptophan solids, thus alleviating the need for 1 H decoupling. [27] …”

Rapid measurement of long-range distances in proteins by multidimensional 13C–19F REDOR NMR under fast magic-angle spinning

“…Recently, we showed that biosynthetic incorporation of a small number of fluorine atoms into proteins allows the measurement of multiple 19 F- 19 F distances to ~1.6 nm (Roos et al 2018). These sparse 19 F spins also serve as probes to dephase the surrounding 13 C and 15 N nuclei through commuting dipole-dipole interactions, and hence should permit the measurement of many 13 C- 19 F and 15 N- 19 F distances.…”