Channelrhodopsin unchained: structure and mechanism of a light-gated cation channel.

The new and vibrant field of optogenetics was founded by the seminal discovery of channelrhodopsin, the first light-gated cation channel. Despite the numerous applications that have revolutionised neurophysiology, the functional mechanism is far from understood on the molecular level. An arsenal of biophysical techniques has been established in the last decades of research on microbial rhodopsins.

Team-based learning increases active engagement and enhances development of teamwork and communication skills in a first-year course for veterinary and animal science undergraduates.

Team-based learning (TBL) was implemented into a first-year course (Principles in Animal Behaviour, Welfare and Ethics) for BSc Veterinary Bioscience (VB) and Animal Science (AS) students. TBL is now used widely in teaching medical students, but has had more limited uptake in veterinary education. This study reports its use over 2 years with cohorts of 126 and 138 students in 2011 and 2012, respectively.

The ability to recognize objects from bottlenose dolphin (Tursiops truncatus) echoes generalizes across multiple orientations in humans and neural networks.

Object constancy, the ability to recognize objects despite changes in orientation, has not been well studied in the auditory modality. Dolphins use echolocation for object recognition, and objects ensonified by dolphins produce echoes that can vary significantly as a function of orientation. In this experiment, human listeners had to classify echoes from objects varying in material, shape, and size that were ensonified with dolphin signals.

Exploring the possible role of Glu286 in CcO by electrostatic energy computations combined with molecular dynamics.

Cytochrome c oxidase (CcO) is a central enzyme in aerobic life catalyzing the conversion of molecular oxygen to water and utilizing the chemical energy to pump protons and establish an electrochemical gradient. Despite intense research, it is not understood how CcO achieves unidirectional proton transport and avoids short circuiting the proton pump. Within this work, we analyzed the potential role of Glu286 as a proton valve.

Hybrid and nonhybrid lipids exert common effects on membrane raft size and morphology.

Nanometer-scale domains in cholesterol-rich model membranes emulate lipid rafts in cell plasma membranes (PMs). The physicochemical mechanisms that maintain a finite, small domain size are, however, not well understood. A special role has been postulated for chain-asymmetric or hybrid lipids having a saturated sn-1 chain and an unsaturated sn-2 chain.

Structural differences between the closed and open states of channelrhodopsin-2 as observed by EPR spectroscopy.

Channelrhodopsin is a cation channel with the unique property of being activated by light. To address structural changes of the open state of the channel, two variants, which contain either 1 or 2 wild-type cysteines, were derivatised with nitroxide spin label and subjected to electron paramagnetic resonance spectroscopy. Both variants contained the C128T mutation to trap the long-lived P3(520) state by illumination.

The [NiFe]-hydrogenase accessory chaperones HypC and HybG of Escherichia coli are iron- and carbon dioxide-binding proteins.

[NiFe]-hydrogenase accessory proteins HypC and HypD form a complex that binds a Fe-(CN)₂CO moiety and CO₂. In this study two HypC homologues from Escherichia coli were purified under strictly anaerobic conditions and both contained sub-stoichiometric amounts of iron (approx. 0.

Surface-enhanced infrared absorption spectroscopy (SEIRAS) to probe monolayers of membrane proteins.

Surface-enhanced infrared absorption spectroscopy (SEIRAS) represents a variation of conventional infrared spectroscopy and exploits the signal enhancement exerted by the plasmon resonance of nano-structured metal thin films. The surface enhancement decays in about 10nm with the distance from the surface and is, thus, perfectly suited to selectively probe monolayers of biomembranes. Peculiar to membrane proteins is their vectorial functionality, the probing of which requires proper orientation within the membrane.

Phase diagram of a 4-component lipid mixture: DSPC/DOPC/POPC/chol.

We report the first 4-component phase diagram for the lipid bilayer mixture, DSPC/DOPC/POPC/chol (distearoylphosphatidylcholine/dioleoylphosphatidylcholine/1-palmitoyl, 2-oleoylphosphatidylcholine/cholesterol). This phase diagram, which has macroscopic Ld+Lo phase domains, clearly shows that all phase boundaries determined for the 3-component mixture containing DOPC transition smoothly into the boundaries for the 3-component mixture containing POPC, which has nanoscopic phase domains of Ld+Lo. Our studies start from two published ternary phase diagrams, and show how these can be combined into a quaternary phase diagram by study of a few hundred samples of intermediate compositions.

HypD is the scaffold protein for Fe-(CN)2CO cofactor assembly in [NiFe]-hydrogenase maturation.

[NiFe]-hydrogenases bind a NiFe-(CN)2CO cofactor in their catalytic large subunit. The iron-sulfur protein HypD and the small accessory protein HypC play a central role in the generation of the CO and CN(-) ligands. Infrared spectroscopy identified signatures on an anaerobically isolated HypCD complex that are reminiscent of those in the hydrogenase active site, suggesting that this complex is the assembly site of the Fe-(CN)2CO moiety of the cofactor prior to its transfer to the hydrogenase large subunit.

Limited perturbation of a DPPC bilayer by fluorescent lipid probes: a molecular dynamics study.

The properties of lipid bilayer nanometer-scale domains could be crucial for understanding cell membranes. Fluorescent probes are often used to study bilayers, yet their effects on host lipids are not well understood. We used molecular dynamics simulations to investigate perturbations in a fluid DPPC bilayer upon incorporation of three indocarbocyanine probes: DiI-C18:0, DiI-C18:2, or DiI-C12:0.

Ultrafast infrared spectroscopy on channelrhodopsin-2 reveals efficient energy transfer from the retinal chromophore to the protein.

The primary reaction dynamics of channelrhodopsin-2 was investigated using femtosecond vis-pump/mid-IR probe spectroscopy. Due to the fast deactivation of the excited state in channelrhodopsin-2, it is possible to observe the direct impact of retinal isomerization on the protein surrounding. We show that the dominant negative band at 1665 cm(-1) tentatively assigned to an amide I vibration is developed with a time constant of 0.

Using small-angle neutron scattering to detect nanoscopic lipid domains.

The cell plasma membrane is a complex system, which is thought to be capable of exhibiting non-random lateral organization. Studies of live cells and model membranes have yielded mechanisms responsible for the formation, growth, and maintenance of nanoscopic heterogeneities, although the existence and mechanisms that give rise to these heterogeneities remain controversial. Small-angle neutron scattering (SANS) is a tool ideally suited to interrogate lateral heterogeneity in model membranes, primarily due to its unique spatial resolution (i.

Transient protonation changes in channelrhodopsin-2 and their relevance to channel gating.

The discovery of the light-gated ion channel channelrhodopsin (ChR) set the stage for the novel field of optogenetics, where cellular processes are controlled by light. However, the underlying molecular mechanism of light-induced cation permeation in ChR2 remains unknown. Here, we have traced the structural changes of ChR2 by time-resolved FTIR spectroscopy, complemented by functional electrophysiological measurements.

Bilayer thickness mismatch controls domain size in model membranes.

The observation of lateral phase separation in lipid bilayers has received considerable attention, especially in connection to lipid raft phenomena in cells. It is widely accepted that rafts play a central role in cellular processes, notably signal transduction. While micrometer-sized domains are observed with some model membrane mixtures, rafts much smaller than 100 nm-beyond the reach of optical microscopy-are now thought to exist, both in vitro and in vivo.

Phase behavior and domain size in sphingomyelin-containing lipid bilayers.

Membrane raft size measurements are crucial to understanding the stability and functionality of rafts in cells. The challenge of accurately measuring raft size is evidenced by the disparate reports of domain sizes, which range from nanometers to microns for the ternary model membrane system sphingomyelin (SM)/1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)/cholesterol (Chol). Using Förster resonance energy transfer (FRET) and differential scanning calorimetry (DSC), we established phase diagrams for porcine brain SM (bSM)/dioleoyl-sn-glycero-3-phosphocholine (DOPC)/Chol and bSM/POPC/Chol at 15 and 25°C.

Polarization-Entangled Photon Pairs From Periodically-Poled Crystalline Waveguides Over a Range of Frequencies.

We propose a method to extend the frequency range of polarization entanglement in periodically poled rubidium-doped potassium titanyl phosphate (Rb:KTP) waveguides. Our calculations predict that output wavelengths from 1130 nm to 1257 nm may be achieved using Rb:KTP by the appropriate selection of a direction of propagation for the waveguide. The fidelity using a poling period of 1 mm is approximately 0.

Interactions between ether phospholipids and cholesterol as determined by scattering and molecular dynamics simulations.

Cholesterol and ether lipids are ubiquitous in mammalian cell membranes, and their interactions are crucial in ether lipid mediated cholesterol trafficking. We report on cholesterol's molecular interactions with ether lipids as determined using a combination of small-angle neutron and X-ray scattering, and all-atom molecular dynamics (MD) simulations. A scattering density profile model for an ether lipid bilayer was developed using MD simulations, which was then used to simultaneously fit the different experimental scattering data.

[NiFe]-hydrogenase maturation: isolation of a HypC-HypD complex carrying diatomic CO and CN- ligands.

The HypC and HypD maturases are required for the biosynthesis of the Fe(CN)(2)CO cofactor in the large subunit of [NiFe]-hydrogenases. Using infrared spectroscopy we demonstrate that an anaerobically purified, Strep-tagged HypCD complex from Escherichia coli exhibits absorption bands characteristic of diatomic CO and CN(-) ligands as well as CO(2). Metal and sulphide analyses revealed that along with the [4Fe-4S](2+) cluster in HypD, the complex has two additional oxygen-labile Fe ions.

Spatiotemporal source analysis in scalp EEG vs. intracerebral EEG and SPECT: a case study in a 2-year-old child.

Objective: This case study aims to demonstrate that spatiotemporal spike discrimination and source analysis are effective to monitor the development of sources of epileptic activity in time and space. Therefore, they can provide clinically useful information allowing a better understanding of the pathophysiology of individual seizures with time- and space-resolved characteristics of successive epileptic states, including interictal, preictal, postictal, and ictal states.

Methods: High spatial resolution scalp EEGs (HR-EEG) were acquired from a 2-year-old girl with refractory central epilepsy and single-focus seizures as confirmed by intracerebral EEG recordings and ictal single-photon emission computed tomography (SPECT).

Model-based approaches for the determination of lipid bilayer structure from small-angle neutron and X-ray scattering data.

Some of our recent work has resulted in the detailed structures of fully hydrated, fluid phase phosphatidylcholine (PC) and phosphatidylglycerol (PG) bilayers. These structures were obtained from the joint refinement of small-angle neutron and X-ray data using the scattering density profile (SDP) models developed by Kučerka et al. (Biophys J 95:2356-2367, 2008; J Phys Chem B 116:232-239, 2012).

Molecular structures of fluid phase phosphatidylglycerol bilayers as determined by small angle neutron and X-ray scattering.

We have determined the molecular structures of commonly used phosphatidylglycerols (PGs) in the commonly accepted biologically relevant fluid phase. This was done by simultaneously analyzing small angle neutron and X-ray scattering data, with the constraint of measured lipid volumes. We report the temperature dependence of bilayer parameters obtained using the one-dimensional scattering density profile model - which was derived from molecular dynamics simulations - including the area per lipid, the overall bilayer thickness, as well as other intrabilayer parameters (e.

Kinetics of proton release and uptake by channelrhodopsin-2.

Electrophysiological experiments showed that the light-activated cation channel channelrhodopsin-2 (ChR2) pumps protons in the absence of a membrane potential. We determined here the kinetics of transient pH change using a water-soluble pH-indicator. It is shown that ChR2 released protons prior to uptake with a stoichiometry of 0.

Scanning near-field IR microscopy of proteins in lipid bilayers.

We use infrared near-field microscopy to chemically map the morphology of biological matrices. The investigated sample is built up from surface-tethered membrane proteins (cytochrome c oxidase) reconstituted in a lipid bilayer. We have carried out infrared near-field measurements in the frequency range between 1600 and 1800 cm(-1).