High-Sensitivity Detection of Nanometer ¹H–¹⁹F Distances for Protein Structure Determination by ¹H-Detected Fast MAS NMR

Abstract

Protein structure determination by solid-state NMR requires the measurement of many interatomic distances through dipole-dipole couplings. To obtain multiple long-range distance restraints rapidly and with high sensitivity, here we demonstrate a new 1 H-detected fast magicangle-spinning (MAS) NMR technique that yields many long distances in a 2D-resolved fashion. The distances are measured up to ~15 Å, with an accuracy of better than 10%, between 1 H and 19 F, two nuclear spins that have the highest gyromagnetic ratios. Exogenous fluorines are sparsely introduced into the aromatic residues of the protein, which is perdeuterated and back-exchanged to give amide protons. This 1 H-19 F distance experiment, termed 2D HSQC-REDOR, is demonstrated on the singly fluorinated model protein, GB1. We extracted 33 distances between 5-19 F-Trp43 and backbone amide protons, using 2D spectral series that were measured in less than 3 days. Combining these 1 H-19 F distance restraints with 13 C-19 F distances and chemical shifts, we calculated a GB1 structure with a backbone RMSD of 1.73 Å from the high-resolution structure. This 1 H-detected 1 H-19 F distance technique promises to provide a highly efficient tool for constraining the three-dimensional structures of proteins and protein-ligand complexes, with not only precise and fast measurements, but also access to truly long-range distances.

Structure and dynamics of the drug-bound bacterial transporter EmrE in lipid bilayers

“…Our structure determination of the EmrE-TPP + complex is enabled by three recent experimental advances: a long-range multiplexed 1 H- 19 F distance measurement technique 32 , fluorinated TPP + , and the slow-exchanging S64V-EmrE 31 . Because of the large magnetic dipole moments of 1 H and 19 F spins, 1 H- 19 F distances up to~2 nm can now be measured using a twodimensional rotational-echo-double-resonance (REDOR) NMR technique 32 . To utilize this technique, we synthesized F 4 -TPP + ( Supplementary Fig.…”

“…The large number of protein-substrate 1 H- 19 F distances ( Supplementary Fig. 10E) 32 , measured in bilayer-bound EmrE, indicates the relative proximities of residues at the binding pocket. The inequivalent substrate position between the two subunits 42 gives insight into the asymmetric protonation of the two E14 residues 13,14 .…”

“…1 H- 19 F distances restrain the structure of the substratebinding pocket. With the 15 N and 1 H chemical shifts assigned, we turned to the 1 H- 19 F REDOR experiment 32 to measure protein-substrate distances. We detected REDOR dephasing in 2D hNH spectra, which exhibit both backbone H N signals and the sidechain indole Hε signals of the important residue W63 (Fig.…”

“…Further, we use a fluorinated TPP + derivative, tetra(4-fluorophenyl) phosphonium (F 4 -TPP + ) ( Fig. 1a), which resembles TPP + in the transport activity, and employ a multidimensional 1 H- 19 F NMR technique 32 to measure a large number of protein-substrate distances. These distances constrain the binding-site structure.…”

Fully hydrophobic HIV gp41 adopts a hemifusion-like conformation in phospholipid bilayers

“…To further test the hemifusion structural model, it will be important to obtain additional long-range distance restraints. These may be obtained from paramagnetically tagged protein or fluorinated protein by exploiting paramagnetic relaxation enhancement or 19 F distance NMR techniques (32)(33)(34)(35), respectively. Finally, mixed labeled protein samples will be important to determine the oligomeric structure of this gp41 hemifusion intermediate.…”