The La-related protein 1-specific domain repurposes HEAT-like repeats to directly bind a 5′TOP sequence

La-related protein 1 (LARP1) regulates the stability of many mRNAs. These include 5′TOPs, mTOR-kinase responsive mRNAs with pyrimidine-rich 5′ UTRs, which encode ribosomal proteins and translation factors. We determined that the highly conserved LARP1-specific C-terminal DM15 region of human LARP1 directly binds a 5′TOP sequence. The crystal structure of this DM15 region refined to 1.86 Å resolution has three structurally related and evolutionarily conserved helix-turn-helix modules within each monomer. These motifs resemble HEAT repeats, ubiquitous helical protein-binding structures, but their sequences are inconsistent with consensus sequences of known HEAT modules, suggesting this structure has been repurposed for RNA interactions. A putative mTORC1-recognition sequence sits within a flexible loop C-terminal to these repeats. We also present modelling of pyrimidine-rich single-stranded RNA onto the highly conserved surface of the DM15 region. These studies lay the foundation necessary for proceeding toward a structural mechanism by which LARP1 links mTOR signaling to ribosome biogenesis

Fluoroalkyl and Alkyl Chains Have Similar Hydrophobicities in Binding to the “Hydrophobic Wall” of Carbonic Anhydrase

This paper describes the performance of junctions based on self-assembled monolayers (SAMs) as the functional element of a half-wave rectifier (a simple circuit that converts, or rectifies, an alternating current (AC) signal to a direct current (DC) signal). Junctions with SAMs of 11-(ferrocenyl)-1-undecanethiol or 11-(biferrocenyl)-1-undecanethiol on ultraflat, template-stripped Ag (AgTS) bottom electrodes, and contacted by top electrodes of eutectic indium–gallium (EGaIn), rectified AC signals, while similar junctions based on SAMs of 1-undecanethiol—SAMs lacking the ferrocenyl terminal group—did not. SAMs in these AC circuits (operating at 50 Hz) remain stable over a larger window of applied bias than in DC circuits. AC measurements, therefore, can investigate charge transport in SAM-based junctions at magnitudes of bias inaccessible to DC measurements. For junctions with SAMs of alkanethiols, combining the results from AC and DC measurements identifies two regimes of bias with different mechanisms of charge transport: (i) low bias (|V| 1.3 V), at which Fowler–Nordheim (FN) tunneling dominates. For junctions with SAMs terminated by Fc moieties, the transition to FN tunneling occurs at |V| ≈ 2.0 V. Furthermore, at sufficient forward bias (V > 0.5 V), hopping makes a significant contribution to charge transport and occurs in series with direct tunneling (V 2.0 V) until FN tunneling activates (V 2.0 V). Thus, for Fc-terminated SAMs at forward bias, three regimes are apparent: (i) direct tunneling (V = 0–0.5 V), (ii) hopping plus direct tunneling (V ≈ 0.5–2.0 V), and (iii) FN tunneling (V 2.0 V). Since hopping does not occur at reverse bias, only two regimes are present over the measured range of reverse bias. This difference in the mechanisms of charge transport at forward and reverse bias for junctions with Fc moieties resulted in large rectification ratios (R > 100) and enabled half-wave rectification.Chemistry and Chemical Biolog

Interactions between Hofmeister Anions and the Binding Pocket of a Protein

This paper uses the binding pocket of human carbonic anhydrase II (HCAII, EC 4.2.1.1) as a tool to examine the properties of Hofmeister anions that determine (i) where, and how strongly, they associate with concavities on the surfaces of proteins and (ii) how, upon binding, they alter the structure of water within those concavities. Results from X-ray crystallography and isothermal titration calorimetry show that most anions associate with the binding pocket of HCAII by forming inner-sphere ion pairs with the Zn2+ cofactor. In these ion pairs, the free energy of anion–Zn2+ association is inversely proportional to the free energetic cost of anion dehydration; this relationship is consistent with the mechanism of ion pair formation suggested by the “law of matching water affinities”. Iodide and bromide anions also associate with a hydrophobic declivity in the wall of the binding pocket. Molecular dynamics simulations suggest that anions, upon associating with Zn2+, trigger rearrangements of water that extend up to 8 Å away from their surfaces. These findings expand the range of interactions previously thought to occur between ions and proteins by suggesting that (i) weakly hydrated anions can bind complementarily shaped hydrophobic declivities, and that (ii) ion-induced rearrangements of water within protein concavities can (in contrast with similar rearrangements in bulk water) extend well beyond the first hydration shells of the ions that trigger them. This study paints a picture of Hofmeister anions as a set of structurally varied ligands that differ in size, shape, and affinity for water and, thus, in their ability to bind to—and to alter the charge and hydration structure of—polar, nonpolar, and topographically complex concavities on the surfaces of proteins.Chemistry and Chemical Biolog

Improvement Of The Crystallizability And Expression Of An Rna Crystallization Chaperone

Crystallizing RNA has been an imperative and challenging task in the world of RNA research. Assistive methods such as chaperone-assisted RNA crystallography (CARC), employing monoclonal antibody fragments (Fabs) as crystallization chaperones have enabled us to obtain RNA crystal structures by forming crystal contacts and providing initial phasing information. Despite the early successes, the crystallization of large RNA-Fab complex remains a challenge in practice. The possible reason for this difficulty is that the Fab scaffold has not been optimized for crystallization in complex with RNA. Here, we have used the surface entropy reduction (SER) technique for the optimization of ΔC209 P4-P6/Fab2 model system. Protruding lysine and glutamate residues were mutated to a set of alanines or serines to construct Fab2SMA or Fab2SMS. Expression with the shake flask approach was optimized to allow large scale production for crystallization. Crystal screening shows that significantly higher crystal-forming ratio was observed for the mutant complexes. As the chosen SER residues are far away from the CDR regions of the Fab, the same set of mutations can now be directly applied to other Fabs binding to a variety of ribozymes and riboswitches to improve the crystallizability of Fab-RNA complex. The Authors 2011. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved2011 © The Authors 2011. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

How Weight Affects the Perceived Spacing between the Thumb and Fingers during Grasping.

We know much about mechanisms determining the perceived size and weight of lifted objects, but little about how these properties of size and weight affect the body representation (e.g. grasp aperture of the hand). Without vision, subjects (n = 16) estimated spacing between fingers and thumb (perceived grasp aperture) while lifting canisters of the same width (6.6cm) but varied weights (300, 600, 900, and 1200 g). Lifts were performed by movement of either the wrist, elbow or shoulder to examine whether lifting with different muscle groups affects the judgement of grasp aperture. Results for perceived grasp aperture were compared with changes in perceived weight of objects of different sizes (5.2, 6.6, and 10 cm) but the same weight (600 g). When canisters of the same width but different weights were lifted, perceived grasp aperture decreased 4.8% [2.2 ‒ 7.4] (mean [95% CI]; P < 0.001) from the lightest to the heaviest canister, no matter how they were lifted. For objects of the same weight but different widths, perceived weight decreased 42.3% [38.2 ‒ 46.4] from narrowest to widest (P < 0.001), as expected from the size-weight illusion. Thus, despite a highly distorted perception of the weight of objects based on their size, we conclude that proprioceptive afferents maintain a reasonably stable perception of the aperture of the grasping hand over a wide range of object weights. Given the small magnitude of this 'weight-grasp aperture' illusion, we propose the brain has access to a relatively stable 'perceptual ruler' to aid the manipulation of different objects

Poor statistical reporting, inadequate data presentation and spin persist despite editorial advice.

The Journal of Physiology and British Journal of Pharmacology jointly published an editorial series in 2011 to improve standards in statistical reporting and data analysis. It is not known whether reporting practices changed in response to the editorial advice. We conducted a cross-sectional analysis of reporting practices in a random sample of research papers published in these journals before (n = 202) and after (n = 199) publication of the editorial advice. Descriptive data are presented. There was no evidence that reporting practices improved following publication of the editorial advice. Overall, 76-84% of papers with written measures that summarized data variability used standard errors of the mean, and 90-96% of papers did not report exact p-values for primary analyses and post-hoc tests. 76-84% of papers that plotted measures to summarize data variability used standard errors of the mean, and only 2-4% of papers plotted raw data used to calculate variability. Of papers that reported p-values between 0.05 and 0.1, 56-63% interpreted these as trends or statistically significant. Implied or gross spin was noted incidentally in papers before (n = 10) and after (n = 9) the editorial advice was published. Overall, poor statistical reporting, inadequate data presentation and spin were present before and after the editorial advice was published. While the scientific community continues to implement strategies for improving reporting practices, our results indicate stronger incentives or enforcements are needed

Structural basis for recognition of S-adenosylhomocysteine by riboswitches

S-adenosyl-(L)-homocysteine (SAH) riboswitches are regulatory elements found in bacterial mRNAs that up-regulate genes involved in the S-adenosyl-(L)-methionine (SAM) regeneration cycle. To understand the structural basis of SAH-dependent regulation by RNA, we have solved the structure of its metabolite-binding domain in complex with SAH. This structure reveals an unusual pseudoknot topology that creates a shallow groove on the surface of the RNA that binds SAH primarily through interactions with the adenine ring and methionine main chain atoms and discriminates against SAM through a steric mechanism. Chemical probing and calorimetric analysis indicate that the unliganded RNA can access bound-like conformations that are significantly stabilized by SAH to direct folding of the downstream regulatory switch. Strikingly, we find that metabolites bearing an adenine ring, including ATP, bind this aptamer with sufficiently high affinity such that normal intracellular concentrations of these compounds may influence regulation of the riboswitch

Experimental set-up.

<p>A, in both experiments 1 and 2, subjects grasped and lifted a range of standard cylindrical canisters of different weights and widths. The canisters were lifted with the right hand with movement at the wrist, elbow or shoulder. Throughout all experiments, the canisters and the subject’s arm were screened from view. B, shows the canisters of different dimensions lifted in both experiments. For experiment 1, the <i>mid-sized</i> test canisters (width 6.6 cm) were used to measure perceived horizontal spacing between the fingers and thumb (<i>perceived grasp aperture</i>) during a lift. These four test canisters ranged in weight from 300g–1200g. The <i>narrow</i> and <i>wide</i> canisters from experiment 2 each weighed 600g and were used as distractors (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127983#sec002" target="_blank">Methods</a>). For experiment 2, the test canisters included the <i>narrow</i>, <i>mid-sized</i> and <i>wide</i> canisters which each weighed 600g and ranged in size from 5.2cm–10cm. During the lift subjects reported the weight, they perceived to be lifting. Two <i>mid-sized</i> canisters from experiment 1 of 300 g and 900 g were used as distractors (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127983#sec002" target="_blank">Methods</a>). C, in experiment 1, subjects reported their perceived grasp aperture using an A3 sized visual chart with 22 numbered horizontal lines of different lengths that represented the grasp aperture of the hand. D, in experiment 2, subjects reported the weight of milk they perceived to be lifting using a coloured vertical scale marked on a one-litre carton with ten 1.9-cm coloured increments, each divided into upper, middle and lower portions for each colour. Each portion represented 33.3 g.</p

Relationship between actual and perceived grasp aperture (experiment 1), and actual and perceived weight (experiment 2).

<p>A, the relationship between actual and perceived grasp aperture for the three canisters of the same weight (600 g) but different width (5.2, 6.6, 10 cm). Data are presented as mean [95%CI]. Actual and perceived grasp aperture were linearly related with a mean <i>R</i>² value of 0.99 [0.99 ‒ 1.00]. The dashed line is the line of identity. On average, grasp aperture was underestimated. B, the relationship between actual and perceived canister weight for the three canisters of the same width (6.6 cm) but different weight (300, 600, 900 g). Actual and perceived weight were linearly related with a mean <i>R</i>² value of 0.98 [0.97 ‒ 0.99]. Data are presented as mean [95% CI] and the dashed line is the line of identity. On average, canister weight was underestimated.</p