Protonation equilibria and pore-opening structure of the dual-histidine influenza B virus M2 transmembrane proton channel from solid-state NMR

The influenza A and B viruses are the primary cause of seasonal flu epidemics. Common to both viruses is the M2 protein, a homotetrameric transmembrane proton channel that acidifies the virion after endocytosis. Although influenza A M2 (AM2) and B M2 (BM2) are functional analogs, they have little sequence homology, except for a conserved HXXXW motif, which is responsible for proton selectivity and channel gating. Importantly, BM2 contains a second titratable histidine, His-27, in the tetrameric transmembrane domain that forms a reverse WXXXH motif with the gating tryptophan. To understand how His-27 affects the proton conduction property of BM2, we have used solid-state NMR to characterize the pH-dependent structure and dynamics of His-27. In cholesterol-containing lipid membranes mimicking the virus envelope, ¹⁵N NMR spectra show that the His-27 tetrad protonates with higher pKa values than His-19, indicating that the solvent-accessible His-27 facilitates proton conduction of the channel by increasing the proton dissociation rates of His-19. AM2 is inhibited by the amantadine class of antiviral drugs, whereas BM2 has no known inhibitors. We measured the N-terminal interhelical separation of the BM2 channel using fluorinated Phe-5. The interhelical ¹⁹F-¹⁹F distances show a bimodal distribution of a short distance of 7 Å and a long distance of 15–20 Å, indicating that the phenylene rings do not block small-molecule entry into the channel pore. These results give insights into the lack of amantadine inhibition of BM2 and reveal structural diversities in this family of viral proton channels. Keywords: influenza virus; ion channel; membrane protein; solid state NMR; structural biolog

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

The ability to simultaneously measure many long-range distances is critical to efficient and accurate determination of protein structures by solid-state NMR (SSNMR). So far, the most common distance constraints for proteins are [subscript 13]C-[subscript 15]N distances, which are usually measured using the rotational-echo double-resonance (REDOR) technique. However, these measurements are restricted to distances of up to ~ 5 Å due to the low gyromagnetic ratios of [subscript 15]N and [subscript 13]C. Here we present a robust 2D [subscript 13]C–[subscript 19]F REDOR experiment to measure multiple distances to ~ 10 Å. The technique targets proteins that contain a small number of recombinantly or synthetically incorporated fluorines. The [subscript 13]C–19F REDOR sequence is combined with 2D [subscript 13]C–[subscript 13]C correlation to resolve multiple distances in highly [subscript 13]C-labeled proteins. We show that, at the high magnetic fields which are important for obtaining well resolved [subscript 13]C spectra, the deleterious effect of the large [subscript 19]F chemical shift anisotropy for REDOR is ameliorated by fast magic-angle spinning and is further taken into account in numerical simulations. We demonstrate this 2D [subscript 13]C–[subscript 13]C resolved [subscript 13]C–[subscript 19]F REDOR technique on [subscript 13]C, [subscript 15]N-labeled GB1. A 5[superscript -19]F-Trp tagged GB1 sample shows the extraction of distances to a single fluorine atom, while a [subscript 3-19]F-Tyr labeled GB1 sample allows us to evaluate the effects of multi-spin coupling and statistical [subscript 19]F labeling on distance measurement. Finally, we apply this 2D REDOR experiment to membrane-bound influenza BM2 transmembrane peptide, and show that the distance between the proton-selective histidine residue and the gating tryptophan residue differs from the distances in the solution NMR structure of detergent-bound BM2. This 2D [subscript 13]C–[subscript 19]F REDOR technique should facilitate SSNMR-based protein structure determination by increasing the measurable distances to the ~ 10 Å range.National Institutes of Health (grant nos. GM066976 and GM088204

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

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 [superscript 19]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. For [superscript 19]F spins with the same isotropic chemical shift but different anisotropic chemical shifts, intermediate MAS frequencies of 15-25 kHz without 1H irradiation accelerate spin exchange. For spectrally resolved [superscript 19]F-[superscript 19]F spin exchange, [superscript 1]H-[superscript 19]F dipolar recoupling significantly speeds up [superscript 19]F-[superscript 19]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[subscript 3] and CF groups. These curves allow [superscript 19]F-[superscript 19]F distances to be extracted from the measured spin exchange rates after taking into account [superscript 19]F chemical shifts. These results demonstrate the robustness of [superscript 19]F spin exchange NMR for distance measurements in a wide range of biological and chemical systems.NIH (grant no. GM066976)German National Academy of Science Leopoldina Postdoctoral Fellowship (grant no. LPDS-2017–14

Renewable Polymers Derived from Limonene

Renewable natural and synthetic basic substances can be used to produce biodegradable polymers. Several methods of the polymerization of terpene limonene have been evaluated. The polymerization methods evaluated are radical polymerization, cationic polymerization and thiol-ene polymerization. The free-radical polymerization of limonene with azobisisobutyronitrile (AIBN) as an initiator was carried out. The cationic polymerization of limonene was carried out using AlCl3 as a catalyst. The copolymerization of limonene with mercaptoethanol, 2-mercaptoethyl ether without an initiator and with an AIBN initiator was studied and it was also shown that polymerization can proceed spontaneously. The resulting compounds were investigated by NMR and FTIR spectroscopy. The values of the molecular weight characteristics of the samples obtained are presented, such as: number-average molecular weight, hydrodynamic radius and characteristic viscosity, depending on the method of production. The coefficients α (molecular shape) in the Mark–Kuhn–Houwink equation are determined according to the established values of the characteristic viscosity. According to the values obtained, the AC molecules in solution have parameters α 0.14 to 0.26, which corresponds to a good solvent and the molecular shape-dense coil

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

The dimeric transporter, EmrE, effluxes polyaromatic cationic drugs in a proton-coupled manner to confer multidrug resistance in bacteria. Although the protein is known to adopt an antiparallel asymmetric topology, its high-resolution drug-bound structure is so far unknown, limiting our understanding of the molecular basis of promiscuous transport. Here we report an experimental structure of drug-bound EmrE in phospholipid bilayers, determined using 19F and 1H solid-state NMR and a fluorinated substrate, tetra(4-fluorophenyl) phosphonium (F4-TPP+). The drug-binding site, constrained by 214 protein-substrate distances, is dominated by aromatic residues such as W63 and Y60, but is sufficiently spacious for the tetrahedral drug to reorient at physiological temperature. F4-TPP+ lies closer to the proton-binding residue E14 in subunit A than in subunit B, explaining the asymmetric protonation of the protein. The structure gives insight into the molecular mechanism of multidrug recognition by EmrE and establishes the basis for future design of substrate inhibitors to combat antibiotic resistance.NIH (Grants GM066976