Structural Basis for pH-gating of the K+ channel TWIK1 at the selectivity filter.

TWIK1 (K2P1.1, KCNK1) is a widely expressed pH-gated two-pore domain K+ channel (K2P) that contributes to cardiac rhythm generation and insulin release from pancreatic beta cells. TWIK1 displays unique properties among K2Ps including low basal activity and inhibition by extracellular protons through incompletely understood mechanisms. Here, we present cryo-EM structures of TWIK1 in lipid nanodiscs at high and low pH that reveal a previously undescribed gating mechanism at the K+ selectivity filter. At high pH, TWIK1 adopts an open conformation. At low pH, protonation of an extracellular histidine results in a cascade of conformational changes that close the channel by sealing the top of the selectivity filter, displacing the helical cap to block extracellular ion access pathways, and opening gaps for lipid block of the intracellular cavity. These data provide a mechanistic understanding for extracellular pH-gating of TWIK1 and illustrate how diverse mechanisms have evolved to gate the selectivity filter of K+ channels.

High-performance microbial opsins for spatially and temporally precise perturbations of large neuronal networks.

The biophysical properties of existing optogenetic tools constrain the scale, speed, and fidelity of precise optogenetic control. Here, we use structure-guided mutagenesis to engineer opsins that exhibit very high potency while retaining fast kinetics. These new opsins enable large-scale, temporally and spatially precise control of population neural activity. We extensively benchmark these new opsins against existing optogenetic tools and provide a detailed biophysical characterization of a diverse family of opsins under two-photon illumination. This establishes a resource for matching the optimal opsin to the goals and constraints of patterned optogenetics experiments. Finally, by combining these new opsins with optimized procedures for holographic photostimulation, we demonstrate the simultaneous coactivation of several hundred spatially defined neurons with a single hologram and nearly double that number by temporally interleaving holograms at fast rates. These newly engineered opsins substantially extend the capabilities of patterned illumination optogenetic paradigms for addressing neural circuits and behavior.

Structural properties of D-mannopyranosyl rings containing O-acetyl side-chains.

The crystal structures of 1,2,3,4,6-penta-O-acetyl-α-D-mannopyranose, C16H22O11, and 2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyl-(1→2)-3,4,6-tri-O-acetyl-α-D-mannopyranosyl-(1→3)-1,2,4,6-tetra-O-acetyl-α-D-mannopyranose, C40H54O27, were determined and compared to those of methyl 2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside, methyl α-D-mannopyranoside and methyl α-D-mannopyranosyl-(1→2)-α-D-mannopyranoside to evaluate the effects of O-acetylation on bond lengths, bond angles and torsion angles. In general, O-acetylation exerts little effect on the exo- and endocyclic C-C and endocyclic C-O bond lengths, but the exocyclic C-O bonds involved in O-acetylation are lengthened by ∼0.02 Å. The conformation of the O-acetyl side-chains is highly conserved, with the carbonyl O atom either eclipsing the H atom attached to a 2°-alcoholic C atom or bisecting the H-C-H bond angle of a 1°-alcoholic C atom. Of the two C-O bonds that determine O-acetyl side-chain conformation, that involving the alcoholic C atom exhibits greater rotational variability than that involving the carbonyl C atom. These findings are in good agreement with recent solution NMR studies of O-acetyl side-chain conformations in saccharides. Experimental evidence was also obtained to confirm density functional theory (DFT) predictions of C-O and O-H bond-length behavior in a C-O-H fragment involved in hydrogen bonding.

O-Benzoyl side-chain conformations in 2,3,4,6-tetra-O-benzoyl-ß-D-galactopyranosyl-(1→4)-1,2,6-tri-O-benzoyl-ß-D-glucopyranose (ethyl acetate solvate) and 1,2,4,6-tetra-O-benzoyl-ß-D-glucopyranose (acetone solvate).

The crystal structures of 2,3,4,6-tetra-O-benzoyl-ß-D-galactopyranosyl-(1→4)-1,2,6-tri-O-benzoyl-ß-D-glucopyranose ethyl acetate hemisolvate, C61H50O18·0.5C4H8O2, and 1,2,4,6-tetra-O-benzoyl-ß-D-glucopyranose acetone monosolvate, C34H28O10·C3H6O, were determined and compared to those of methyl ß-D-galactopyranosyl-(1→4)-ß-D-glucopyranoside (methyl ß-lactoside) and methyl ß-D-glucopyranoside hemihydrate, C7H14O6·0.5H2O, to evaluate the effects of O-benzoylation on bond lengths, bond angles and torsion angles. In general, O-benzoylation exerts little effect on exo- and endocyclic C-C and endocyclic C-O bond lengths, but exocyclic C-O bonds involved in O-benzoylation are lengthened by 0.02-0.04 Å depending on the site of substitution. The conformation of the O-benzoyl side-chains is highly conserved, with the carbonyl O atom either eclipsing the H atom attached to a 2°-alcoholic C atom or bisecting the H-C-H bond angle of an 1°-alcoholic C atom. Of the three bonds that determine the side-chain geometry, the C-O bond involving the alcoholic C atom exhibits greater rotational variability than the remaining C-O and C-C bonds involving the carbonyl C atom. These findings are in good agreement with recent solution NMR studies of the O-acetyl side-chain conformation in saccharides.

Enzymatic synthesis of ribo- and 2'-deoxyribonucleosides from glycofuranosyl phosphates: An approach to facilitate isotopic labeling.

Milligram quantities of α-D-ribofuranosyl 1-phosphate (sodium salt) (αR1P) were prepared by the phosphorolysis of inosine, catalyzed by purine nucleoside phosphorylase (PNPase). The αR1P was isolated by chromatography in >95% purity and characterized by 1H and 13C NMR spectroscopy. Aqueous solutions of αR1P were stable at pH 6.4 and 4 °C for several months. The isolated αR1P was N-glycosylated with different nitrogen bases (adenine, guanine and uracil) using PNPase or uridine phosphorylase (UPase) to give the corresponding ribonucleosides in high yield based on the glycosyl phosphate. This methodology is attractive for the preparation of stable isotopically labeled ribo- and 2'-deoxyribonucleosides because of the ease of product purification and convenient use and recycling of nitrogen bases. The approach eliminates the need for separate reactions to prepare individual furanose-labeled ribonucleosides, since only one ribonucleoside (inosine) needs to be labeled, if desired, in the furanose ring, the latter achieved by a high-yield chemical N-glycosylation. 2'-Deoxyribonucleosides were prepared from 2'-deoxyinosine using the same methodology with minor modifications.

Conformational Populations of ß-(1→4) O-Glycosidic Linkages Using Redundant NMR J-Couplings and Circular Statistics.

Twelve disaccharides containing ß-(1→4) linkages and displaying systematic structural variations in the vicinity of these linkages were selectively labeled with 13C to facilitate measurements of multiple NMR spin-spin (scalar; J) coupling constants (JCH and JCC values) across their O-glycosidic linkages. Ensembles of spin-couplings (2JCOC, 3JCOCH, 3JCOCC) sensitive to the two linkage torsion angles, phi (ϕ) and psi (ψ), were analyzed by using parametrized equations obtained from density functional theory (DFT) calculations, Fredholm theory, and circular statistics to calculate experiment-based rotamer populations for ϕ and ψ in each disaccharide. With the statistical program MA'AT, torsion angles ϕ and ψ were modeled as a single von Mises distribution, which yielded two parameters, the mean position and the circular standard deviation (CSD) for each angle. The NMR-derived rotamer populations were compared to those obtained from 1 µs aqueous molecular dynamics (MD) simulations and crystallographic database statistical analyses. Conformer populations obtained exclusively from the MA'AT treatment of redundant J-couplings were in very good agreement with those obtained from the MD simulations, providing evidence that conformational populations can be determined by NMR for mobile molecular elements such as O-glycosidic linkages with minimal input from theory. The approach also provides an experimental means to validate the conformational preferences predicted from MD simulations. The conformational behaviors of ϕ in the 12 disaccharides were very similar, but those of ψ varied significantly, allowing a classification of the 12 disaccharides based on preferred linkage conformation in solution.

O-Acetyl Side-Chains in Monosaccharides: Redundant NMR Spin-Couplings and Statistical Models for Acetate Ester Conformational Analysis.

α- and ß-d-glucopyranose monoacetates 1-3 were prepared with selective 13C enrichment in the O-acetyl side-chain, and ensembles of 13C-1H and 13C-13C NMR spin-couplings (J-couplings) were measured involving the labeled carbons. Density functional theory (DFT) was applied to a set of model structures to determine which J-couplings are sensitive to rotation of the ester bond θ. Eight J-couplings (1JCC, 2JCH, 2JCC, 3JCH, and 3JCC) were found to be sensitive to θ, and four equations were parametrized to allow quantitative interpretations of experimental J-values. Inspection of J-coupling ensembles in 1-3 showed that O-acetyl side-chain conformation depends on molecular context, with flanking groups playing a dominant role in determining the properties of θ in solution. To quantify these effects, ensembles of J-couplings containing four values were used to determine the precision and accuracy of several 2-parameter statistical models of rotamer distributions across θ in 1-3. The statistical method used to generate these models has been encoded in a newly developed program, MA'AT, which is available for public use. These models were compared to O-acetyl side-chain behavior observed in a representative sample of crystal structures, and in molecular dynamics (MD) simulations of O-acetylated model structures. While the functional form of the model had little effect on the precision of the calculated mean of θ in 1-3, platykurtic models were found to give more precise estimates of the width of the distribution about the mean (expressed as circular standard deviations). Validation of these 2-parameter models to interpret ensembles of redundant J-couplings using the O-acetyl system as a test case enables future extension of the approach to other flexible elements in saccharides, such as glycosidic linkage conformation.