Generation and evaluation of a large mutational library from the Escherichia coli mechanosensitive channel of large conductance, MscL - Implications for channel gating and evolutionary design

Random mutagenesis of the mechanosensitive channel of large conductance (MscL) from Escherichia coli coupled with a high-throughput functional screen has provided new insights into channel structure and function. Complementary interactions of conserved residues proposed in a computational model for gating have been evaluated, and important functional regions of the channel have been identified. Mutational analysis shows that the proposed S1 helix, despite having several highly conserved residues, can be heavily mutated without significantly altering channel function. The pattern of mutations that make MscL more difficult to gate suggests that MscL senses tension with residues located near the lipid headgroups of the bilayer. The range of phenotypical changes seen has implications for a proposed model for the evolutionary origin of mechanosensitive channels

New Views of Multi-Ion Channels

Thus, most site-directed mutagenesis data render it untenable to consider that two or more roughly equivalent high affinity sites govern selectivity in multi-ion pores. The papers by Dang and McCleskey and Kiss et al. respond to this challenge by showing that a model with a single high affinity site, flanked by two binding sites of lower affinity close to the pore entrances, can generate much of the classical multi-ion behavior. The sites need not interact, and the two flanking sites could arise from one of several mechanisms: a featureless charged vestibule, a dehydration step, or a specific weak binding site. The multi-ion pore remains a cornerstone of permeation theory, but the new theory features only a single high affinity site and no mutual repulsion. The high flux rate occurs because ions pause at the flanking sites and reequilibrate thermally, gaining enough energy to move over the next barrier

Room-Temperature Alternative to the Arbuzov Reaction: The Reductive Deoxygenation of Acyl Phosphonates

The reductive deoxygenation of acyl phosphonates using a Wolff−Kishner-like sequence is described. This transformation allows direct access to alkyl phosphonates from acyl phosphonates at room temperature. The method can be combined with acyl phosphonate synthesis into a one pot, four-step procedure for the conversion of carboxylic acids into alkyl phosphonates. The methodology works well for a variety of aliphatic acids and shows a functional group tolerance similar to that of other hydrazone-forming reactions

Preparation of Translationally Competent tRNA by Direct Chemical Acylation

Nonsense codon suppression for unnatural amino acid incorporation requires the preparation of a suppressor aminoacyl-tRNA. Chemical acylation strategies are general but inefficient and arduous. A recent report (J. Am. Chem. Soc. 2007, 129, 15848) showed acylation of RNA mediated by lanthanum(III) using amino acid phosphate esters. The successful implementation of this methodology to full-length suppressor tRNA is described, and it is shown that the derived aminoacyl-tRNA is translationally competent in Xenopus oocytes

Characterization of Saturn's bow shock: magnetic field observations of quasi-perpendicular shocks

Collisionless shocks vary drastically from terrestrial to astrophysical regimes resulting in radically different characteristics. This poses two complexities. First, separating the influences of these parameters on physical mechanisms such as energy dissipation. Second, correlating observations of shock waves over a wide range of each parameter, enough to span across different regimes. Investigating the latter has been restricted since the majority of studies on shocks at exotic regimes (such as supernova remnants) have been achieved either remotely or via simulations, but rarely by means of in situ observations. Here we present the parameter space of MA bow shock crossings from 2004 to 2014 as observed by the Cassini spacecraft. We find that Saturn's bow shock exhibits characteristics akin to both terrestrial and astrophysical regimes (MA of order 100), which is principally controlled by the upstream magnetic field strength. Moreover, we determined the θBn of each crossing to show that Saturn's (dayside) bow shock is predominantly quasi-perpendicular by virtue of the Parker spiral at 10 AU. Our results suggest a strong dependence on MA in controlling the onset of physical mechanisms in collisionless shocks, particularly nontime stationarity and variability. We anticipate that our comprehensive assessment will yield deeper insight into high MA collisionless shocks and provide a broader scope for understanding the structures and mechanisms of collisionless shocks

Establishing an Ion Pair Interaction in the Homomeric {rho}1 {gamma}-Aminobutyric Acid Type A Receptor That Contributes to the Gating Pathway

{gamma}-Aminobutyric acid type A (GABAA) receptors are members of the Cys-loop superfamily of ligand-gated ion channels. Upon agonist binding, the receptor undergoes a structural transition from the closed to the open state, but the mechanism of gating is not well understood. Here we utilized a combination of conventional mutagenesis and the high precision methodology of unnatural amino acid incorporation to study the gating interface of the human homopentameric {rho}1 GABAA receptor. We have identified an ion pair interaction between two conserved charged residues, Glu92 in loop 2 of the extracellular domain and Arg258 in the pre-M1 region. We hypothesize that the salt bridge exists in the closed state by kinetic measurements and free energy analysis. Several other charged residues at the gating interface are not critical to receptor function, supporting previous conclusions that it is the global charge pattern of the gating interface that controls receptor function in the Cys-loop superfamily

Subunit Stoichiometry of a Heteromultimeric G protein-coupled Inward-rectifier K^+ Channel

We investigated the stoichiometry of the heteromultimeric G protein-coupled inward-recitfier K^+ channel (GIRK) formed from GIRK1 and GIRK4 subunits. Multimeric GIRK constructs with several concatenated channel subunits were expressed in Xenopus oocytes. Coexpression of various trimeric constructs with different monomers clearly showed that the functional channel has stoichiometry (GIRK1)_2(GIRK4)_2. Efforts to establish a preferred arrangement of subunits around the channel pore suggest that more than one arrangement may be viable

A Stereochemical Test of a Proposed Structural Feature of the Nicotinic Acetylcholine Receptor

Understanding the gating mechanism of the nicotinic acetylcholine receptor (nAChR) and similar channels constitutes a significant challenge in chemical neurobiology. In the present work, we use a stereochemical probe to evaluate a proposed pin-into-hydrophobic socket mechanism for the αVal46 side chain of the nAChR. Utilizing nonsense suppression methodology we incorporated isoleucine (Ile), O-methyl threonine (Omt) and threonine (Thr) as well as their side chain epimers (the allo counterparts). Surprisingly, our results indicate that only the pro-S methyl group of the αVal46 side chain is sensitive to changes in hydrophobicity, consistent with the precise geometrical requirements of the pin-into-socket mechanism

Channeled propagation of solar particles

Bartley (1966) and McCracken and Ness (1966) identified bundles of interplanetary magnetic field (IMF) lines that differed in direction from the interplanetary field lines in which they were imbedded. These bundles, called filaments differed in direction by as much as several tens of degrees from the surrounding field. The filaments werre first noticed due to the large and sudden change in flow direction of highly anisotropic solar flare protons in the energy range 1 to 13 MeV. Passage of the filaments over the spacecraft required a few hours, implying a diameter for the filaments of approximately 3 x 10 to the 6th power km at a distance of 1 AU from the Sun. In 1968, Jakipii and Parker used Leighton's hypothesis of random walk of magnetic field lines associated with granules and supergranules (1964) to develop a picture of an interplanetary medium composed of a tangle of field lines frozen into the solar wind, but whose feet were carried about by the random motions at the solar surface. Jakipii and Parker noted that using a correlation length of 15,000 km - about the radius of a supergranule - the magnetic structure would be 3 x 10 to the 6th power km in size of the filaments as determined by Bartley and McCracken and Ness. These workers did not find changes in the solar particle intensity, anisotropy ratio or energy spectrum as the spacecraft entered the filament