¹⁹F‐MAS NMR on Proteorhodopsin: Enhanced Protocol for Site‐Specific Labeling for General Application to Membrane Proteins^†

Abstract

Proteorhodopsin (PR) is a light-driven proton pump found in near-surface marine gamma-proteobacteria. The green absorbing variant has three cysteines at positions 107, 156 and 175. We probed the accessibility of these residues by (19)F-MAS NMR. For this purpose, an efficient but simple protocol for chemical fluorine labeling of accessible cysteines in membrane proteins was established. This one-step reaction was applied to detergent-solubilized PR before reconstitution into phospholipids. All three cysteines could be labeled and showed distinct (19)F chemical shifts with different integral intensities. The accessibility of these cysteines is discussed in the context of a homology model. With the chemical cysteine labeling procedure shown here, an attractive option for site-directed solid-state NMR studies on other membrane proteins is offered due to the high intrinsic sensitivity of (19)F-MAS NMR.

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

“…[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. [25] 19 F NMR has been applied to investigate proteins, lipids, nucleic acids, synthetic small-molecule ligands, as well as their complexes, both in solution [16, 21] and in the solid state.…”

“…[25] 19 F NMR has been applied to investigate proteins, lipids, nucleic acids, synthetic small-molecule ligands, as well as their complexes, both in solution [16, 21] and in the solid state. [22–24] 19 F MAS NMR of biological systems remains underutilized, due to challenges associated with inherently broad lines due to strong homonuclear 19 F- 19 F and heteronuclear 19 F- 1 H dipolar couplings, particularly at traditionally used MAS frequencies below 25 kHz. Only three recent studies used faster spinning frequencies.…”

Permeating disciplines: Overcoming barriers between molecular simulations and classical structure-function approaches in biological ion transport

“…Finally, certain nuclei with high NMR sensitivity—such as 19 F—occur rarely in proteins but often in drugs, providing excellent reporters to probe solvent accessibility, conformational dynamics, and/or binding in ion transport and related processes [e.g. 55–57]. In one example, molecular dynamics simulations were used to predict binding sites for the fluorinated general anesthetic isoflurane in the voltage-gated sodium channel NaChBac, and the identified residues were 19 F-labeled.…”

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

“…[13,14] Fluorine is absent from any naturally occurring biological molecule,y et it can be readily and selectively incorporated into proteins, [15][16][17] largely without causing major structural perturbations. [18] 19 FNMR spectroscopy,both solution and solid-state,has therefore emerged as an essential method with broad applications in pharmaceutical chemistry (~30 %ofall drugs at present in the clinic contain fluorine), [19] chemical biology, [20] biochemistry, [16,[21][22][23][24] and materials science. [25] 19 FNMR spectroscopy has been used to investigate proteins,lipids,nucleic acids,and synthetic small-molecule ligands,a sw ell as their complexes,b oth in solution [16,21] and in the solid state.…”

“…[25] 19 FNMR spectroscopy has been used to investigate proteins,lipids,nucleic acids,and synthetic small-molecule ligands,a sw ell as their complexes,b oth in solution [16,21] and in the solid state. [22][23][24] 19 FM AS NMR spectroscopy of biological systems remains underutilized owing to challenges associated with inherently broad lines due to strong homonuclear 19 F- 19 Fand heteronuclear 19 F-1 H dipolar couplings,p articularly at traditionally used MAS frequencies below 25 kHz. Only three recent studies used faster spinning frequencies.Intwo of these studies,resolution enhancements were observed at 35 and 40 kHz, [14,26] whereas areport from our research group demonstrated that frequencies of 40-60 kHz yielded significant line narrowing for fluoro-substituted tryptophan solids,t hus alleviating the need for 1 Hdecoupling.…”