Voltage Sensing in Bacterial Protein Translocation

The bacterial channel SecYEG efficiently translocates both hydrophobic and hydrophilic proteins across the plasma membrane. Translocating polypeptide chains may dislodge the plug, a half helix that blocks the permeation of small molecules, from its position in the middle of the aqueous translocation channel. Instead of the plug, six isoleucines in the middle of the membrane supposedly seal the channel, by forming a gasket around the translocating polypeptide. However, this hypothesis does not explain how the tightness of the gasket may depend on membrane potential. Here, we demonstrate voltage-dependent closings of the purified and reconstituted channel in the presence of ligands, suggesting that voltage sensitivity may be conferred by motor protein SecA, ribosomes, signal peptides, and/or translocating peptides. Yet, the presence of a voltage sensor intrinsic to SecYEG was indicated by voltage driven closure of pores that were forced-open either by crosslinking the plug to SecE or by plug deletion. We tested the involvement of SecY’s half-helix 2b (TM2b) in voltage sensing, since clearly identifiable gating charges are missing. The mutation L80D accelerated voltage driven closings by reversing TM2b’s dipolar orientation. In contrast, the L80K mutation decelerated voltage induced closings by increasing TM2b’s dipole moment. The observations suggest that TM2b is part of a larger voltage sensor. By partly aligning the combined dipole of this sensor with the orientation of the membrane-spanning electric field, voltage may drive channel closure

ANT1 Activation and Inhibition Patterns Support the Fatty Acid Cycling Mechanism for Proton Transport

Adenine nucleotide translocase (ANT) is a well-known mitochondrial exchanger of ATP against ADP. In contrast, few studies have shown that ANT also mediates proton transport across the inner mitochondrial membrane. The results of these studies are controversial and lead to different hypotheses about molecular transport mechanisms. We hypothesized that the H+-transport mediated by ANT and uncoupling proteins (UCP) has a similar regulation pattern and can be explained by the fatty acid cycling concept. The reconstitution of purified recombinant ANT1 in the planar lipid bilayers allowed us to measure the membrane current after the direct application of transmembrane potential ΔΨ, which would correspond to the mitochondrial states III and IV. Experimental results reveal that ANT1 does not contribute to a basal proton leak. Instead, it mediates H+ transport only in the presence of long-chain fatty acids (FA), as already known for UCPs. It depends on FA chain length and saturation, implying that FA’s transport is confined to the lipid-protein interface. Purine nucleotides with the preference for ATP and ADP inhibited H+ transport. Specific inhibitors of ATP/ADP transport, carboxyatractyloside or bongkrekic acid, also decreased proton transport. The H+ turnover number was calculated based on ANT1 concentration determined by fluorescence correlation spectroscopy and is equal to 14.6 ± 2.5 s−1. Molecular dynamic simulations revealed a large positively charged area at the protein/lipid interface that might facilitate FA anion’s transport across the membrane. ANT’s dual function—ADP/ATP and H+ transport in the presence of FA—may be important for the regulation of mitochondrial membrane potential and thus for potential-dependent processes in mitochondria. Moreover, the expansion of proton-transport modulating drug targets to ANT1 may improve the therapy of obesity, cancer, steatosis, cardiovascular and neurodegenerative diseases

A New Theory about Interfacial Proton Diffusion Revisited: The Commonly Accepted Laws of Electrostatics and Diffusion Prevail

The high propensity of protons to stay at interfaces has attracted much attention over the decades. It enables long-range interfacial proton diffusion without relying on titratable residues or electrostatic attraction. As a result, various phenomena manifest themselves, ranging from spillover in material sciences to local proton circuits between proton pumps and ATP synthases in bioenergetics. In an attempt to replace all existing theoretical and experimental insight into the origin of protons’ preference for interfaces, TELP, the “Transmembrane Electrostatically-Localized Protons” hypothesis, has been proposed. The TELP hypothesis envisions static H+ and OH− layers on opposite sides of interfaces that are up to 75 µm thick. Yet, the separation at which the electrostatic interaction between two elementary charges is comparable in magnitude to the thermal energy is more than two orders of magnitude smaller and, as a result, the H+ and OH− layers cannot mutually stabilize each other, rendering proton accumulation at the interface energetically unfavorable. We show that (i) the law of electroneutrality, (ii) Fick’s law of diffusion, and (iii) Coulomb’s law prevail. Using them does not hinder but helps to interpret previously published experimental results, and also helps us understand the high entropy release barrier enabling long-range proton diffusion along the membrane surface

Interfacial water molecules at biological membranes: Structural features and role for lateral proton diffusion

Proton transport at water/membrane interfaces plays a fundamental role for a myriad of bioenergetic processes. Here we have performed ab initio molecular dynamics simulations of proton transfer along two phosphatidylcholine bilayers. As found in previous theoretical studies, the excess proton is preferably located at the water/membrane interface. Further, our simulations indicate that it interacts not only with phosphate head groups, but also with water molecules at the interfaces. Interfacial water molecules turn out to be oriented relative to the lipid bilayers, consistently with experimental evidence. Hence, the specific water-proton interaction may help explain the proton mobility experimentally observed at the membrane interface

The Bacterial Translocon SecYEG Opens upon Ribosome Binding

In co-translational translocation, the ribosome funnel and the channel of the protein translocation complex SecYEG are aligned. For the nascent chain to enter the channel immediately after synthesis, a yet unidentified signal triggers displacement of the SecYEG sealing plug from the pore. Here, we show that ribosome binding to the resting SecYEG channel triggers this conformational transition. The purified and reconstituted SecYEG channel opens to form a large ion-conducting channel, which has the conductivity of the plug deletion mutant. The number of ion-conducting channels inserted into the planar bilayer per fusion event roughly equals the number of SecYEG channels counted by fluorescence correlation spectroscopy in a single proteoliposome. Thus, the open probability of the channel must be close to unity. To prevent the otherwise lethal proton leak, a closed post-translational conformation of the SecYEG complex bound to a ribosome must exist

Interfacial water molecules at biological membranes: Structural features and role for lateral proton diffusion - Fig 8

<p>Top: 2D histogram of the angle between water dipole moment and interface normal, and the distance from the instantaneous water/membrane interface for DOPC-2 system. Trajectories were collected using <i>ab initio</i> MD simulations; Bottom: Schematic representation of approximately 150° orientation of water molecule with respect to the interface.</p

EEG and fMRI Correlates of Insight: A Pilot Study

Insight is described as the sudden solution of a problem and is contrasted with an analytical, step-by-step approach. Traditionally, insight is thought to be associated with activity of the right hemisphere, whereas analytical solutions are thought to be associated with activity of the left hemisphere. However, empirical evidence as to the localization of insight-related brain activity is mixed and inconclusive. Some studies seem to confirm the traditional view, whereas others do not. Moreover, results of EEG and fMRI studies frequently contradict each other. In this study, EEG and fMRI data were recorded while subjects performed the remote association test and for each solved problem were asked to report whether the solution was reached analytically or insightfully. The data were analyzed in a 16-second fragment preceding the subject’s response. Source localization techniques were used in the analysis of EEG data. Based on EEG data, insightful as compared to analytical problem solving was accompanied by high-frequency synchronization in semantic cortical areas of the left hemisphere 10–12 s before the subject’s response. Based on fMRI data, however, insightful solutions were accompanied by increased activity in frontal and temporal regions of the right hemisphere. The results are interpreted in terms of different cognitive processes involved in insightful problem solving, which could be differently reflected in EEG and fMRI data

Schematic of three consecutive proton transfer coordinates.

<p>Oxygen ad hydrogen atoms are shown as red and white spheres, respectively. Hydrogen bonds are represented as dashed lines. <i>v</i>_<i>1</i>, <i>v</i>_<i>2</i> and <i>v</i>_<i>3</i> are the proton transfer coordinates of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193454#pone.0193454.e001" target="_blank">Eq (1)</a>.</p

2D histograms of proton transfer coordinates of DOPC-2 from <i>ab intio</i> MD simulations.

<p>Note that the color bar is on a logarithmic scale. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193454#sec002" target="_blank">Methods</a> section and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193454#pone.0193454.g002" target="_blank">Fig 2</a> and for the definition of proton transfer coordinates.</p