Effective pore size and radius of capture for K+ ions in K-channels

Indexación: Web of Science; Scopus.Reconciling protein functional data with crystal structure is arduous because rare conformations or crystallization artifacts occur. Here we present a tool to validate the dimensions of open pore structures of potassium-selective ion channels. We used freely available algorithms to calculate the molecular contour of the pore to determine the effective internal pore radius (r(E)) in several K-channel crystal structurss. r(E) was operationally defined as the radius of the biggest sphere able to enter the pore from the cytosolic side. We obtained consistent r(E) estimates for MthK and Kv1.2/2.1 structures, with r(E) = 5.3-5.9 angstrom and r(E) = 4.5-5.2 angstrom, respectively. We compared these structural estimates with functional assessments of the internal mouth radii of capture (r(C)) for two electrophysiological counterparts, the large conductance calcium activated K-channel (r(C) = 2.2 angstrom) and the Shaker K-v-channel (r(C) = 0.8 angstrom), for MthK and Kv1.2/2.1 structures, respectively. Calculating the difference between r(E) and r(C), produced consistent size radii of 3.1-3.7 angstrom and 3.6-4.4 angstrom for hydrated K+ ions. These hydrated K+ estimates harmonize with others obtained with diverse experimental and theoretical methods. Thus, these findings validate MthK and the Kv1.2/2.1 structures as templates for open BK and Kv-channels, respectively.http://recursosbiblioteca.unab.cl:2226/articles/srep1989

Gating-induced large aqueous volumetric remodeling and aspartate tolerance in the voltage sensor domain of Shaker K+ channels

Indexación: Scopus.ACKNOWLEDGMENTS. We thank Chris Lingle and Yu Zhou (Washington University) for critical reading of the manuscript and Victoria Prado for Xenopus care and oocyte preparation. We also thank Millennium Scientific Initiative P029-022-F. This work was supported by Fondecyt Postdoctoral Grants 3170599 (to I.D.-F.) and 3160321 (to H.M.).Neurons encode electrical signals with critically tuned voltage-gated ion channels and enzymes. Dedicated voltage sensor domains (VSDs) in these membrane proteins activate coordinately with an unresolved structural change. Such change conveys the transmembrane translocation of four positively charged arginine side chains, the voltage-sensing residues (VSRs; R1–R4). Countercharges and lipid phosphohead groups likely stabilize these VSRs within the low-dielectric core of the protein. However, the role of hydration, a sign-independent charge stabilizer, remains unclear. We replaced all VSRs and their neighboring residues with negatively charged aspartates in a voltage-gated potassium channel. The ensuing mild functional effects indicate that hydration is also important in VSR stabilization. The voltage dependency of the VSR aspartate variants approached the expected arithmetic summation of charges at VSR positions, as if negative and positive side chains faced similar pathways. In contrast, aspartates introduced between R2 and R3 did not affect voltage dependence as if the side chains moved outside the electric field or together with it, undergoing a large displacement and volumetric remodeling. Accordingly, VSR performed osmotic work at both internal and external aqueous interfaces. Individual VSR contributions to volumetric works approached arithmetical additivity but were largely dissimilar. While R1 and R4 displaced small volumes, R2 and R3 volumetric works were massive and vectorially opposed, favoring large aqueous remodeling during VSD activation. These diverse volumetric works are, at least for R2 and R3, not compatible with VSR translocation across a unique stationary charge transfer center. Instead, VSRs may follow separated pathways across a fluctuating low-dielectric septum. © National Academy of Sciences. All rights reserved.https://www.pnas.org/content/115/32/820

Naturally Segregating Variation at Ugt86Dd Contributes to Nicotine Resistance in Drosophila melanogaster

Identifying the sequence polymorphisms underlying complex trait variation is a key goal of genetics research, since knowing the precise causative molecular events allows insight into the pathways governing trait variation. Genetic analysis of complex traits in model systems regularly starts by constructing QTL maps, but generally fails to identify causative sequence polymorphisms. Previously we mapped a series of QTL contributing to resistance to nicotine in a Drosophila melanogaster multiparental mapping resource and here use a battery of functional tests to resolve QTL to the molecular level. One large-effect QTL resided over a cluster of UDP-glucuronosyltransferases, and quantitative complementation tests using deficiencies eliminating subsets of these detoxification genes revealed allelic variation impacting resistance. RNAseq showed that Ugt86Dd had significantly higher expression in genotypes that are more resistant to nicotine, and anterior midgut-specific RNA interference (RNAi) of this gene reduced resistance. We discovered a segregating 22-bp frameshift deletion in Ugt86Dd, and accounting for the InDel during mapping largely eliminates the QTL, implying the event explains the bulk of the effect of the mapped locus. CRISPR/Cas9 editing of a relatively resistant genotype to generate lesions in Ugt86Dd that recapitulate the naturally occurring putative loss-of-function allele, leads to a large reduction in resistance. Despite this major effect of the deletion, the allele appears to be very rare in wild-caught populations and likely explains only a small fraction of the natural variation for the trait. Nonetheless, this putatively causative coding InDel can be a launchpad for future mechanistic exploration of xenobiotic detoxification

Asymmetrical flow field-flow fractionation in purification of an enveloped bacteriophage φ 6

Basic and applied virus research requires specimens that are purified to high homogeneity. Thus, there is much interest in the efficient production and purification of viruses and their subassemblies. Advances in the production steps have shifted the bottle neck of the process to the purification. Nonetheless, the development of purification techniques for different viruses is challenging due to the complex biological nature of the infected cell cultures as well as the biophysical and -chemical differences in the virus particles. We used bacteriophage phi 6 as a model virus in our attempts to provide a new purification method for enveloped viruses. We compared asymmetrical flow field-flow fractionation (AF4)-based virus purification method to the well-established ultracentrifugation-based purification of phi 6. In addition, binding of phi 6 virions to monolithic anion exchange columns was tested to evaluate their applicability in concentrating the AF4 purified specimens. Our results show that AF4 enables one-hour purification of infectious enveloped viruses with specific infectivity of similar to 1 x 10(13) PFU/mg of protein and similar to 65-95% yields. Obtained purity was comparable with that obtained using ultracentrifugation, but the yields from AF4 purification were 2-3-fold higher. Importantly, high quality virus preparations could be obtained directly from crude cell lysates. Furthermore, when used in combination with inline light scattering detectors, AF4 purification could be coupled to simultaneous quality control of obtained virus specimen.Peer reviewe

Charge Ordering in Organic ET Compounds

The charge ordering phenomena in quasi two-dimensional 1/4-filled organic compounds (ET)_2X (ET=BEDT-TTF) are investigated theoretically for the $\theta$ and $\alpha$-type structures, based on the Hartree approximation for the extended Hubbard models with both on-site and intersite Coulomb interactions. It is found that charge ordered states of stripe-type are stabilized for the relevant values of Coulomb energies, while the spatial pattern of the stripes sensitively depends on the anisotropy of the models. By comparing the results of calculations with the experimental facts, where the effects of quantum fluctuation is incorporated by mapping the stripe-type charge ordered states to the S=1/2 Heisenberg Hamiltonians, the actual charge patterns in the insulating phases of $\theta$-(ET)_2MM'(SCN)_4 and $\alpha$-(ET)_2I_3 are deduced. Furthermore, to obtain a unified view among the $\theta$, $\alpha$ and $\kappa$-(ET)_2X families, the stability of the charge ordered state in competition with the dimeric antiferromagnetic state viewed as the Mott insulating state, which is typically realized in $\kappa$-type compounds, and with the paramagnetic metallic state, is also pursued by extracting essential parameters.Comment: 35 pages, 27 figures, submitted to J. Phys. Soc. Jp

High field magnetic resonant properties of beta'-(ET)2SF5CF2SO3

A systematic electron spin resonance (ESR) investigation of the low temperature regime for the (ET)2SF5CF2SO3 system was performed in the frequency range of ~200-700 GHz, using backward wave oscillator sources, and at fields up to 25 T. Newly acquired access to the high frequency and fields shows experimental ESR results in agreement with the nuclear magnetic resonance (NMR) investigation, revealing evidence that the transition seen at 20 K is not of conventional spin-Peierls order. A significant change of the spin resonance spectrum in beta'-(ET)2SF5CF2SO3 at low temperatures, indicates a transition into a three-dimensional-antiferromagnetic (3D AFM) phase.Comment: 4 pages, 7 figures, minor grammatical change

Economic Returns From Grain Sorghum Fed to Steer Calves on Dryland Farms of the High Plains.

16 p