scholarly journals Unifying photocycle model for light adaptation and temporal evolution of cation conductance in channelrhodopsin-2

2019 ◽  
Vol 116 (19) ◽  
pp. 9380-9389 ◽  
Author(s):  
Jens Kuhne ◽  
Johannes Vierock ◽  
Stefan Alexander Tennigkeit ◽  
Max-Aylmer Dreier ◽  
Jonas Wietek ◽  
...  
Keyword(s):  
Rational Design ◽  
Open Channel ◽  
Light Adaptation ◽  
Ion Selectivity ◽  
Continuous Illumination ◽  
Channel State ◽  
Time Resolved ◽  
Open Questions ◽  
Cation Conductance ◽  
High Proton

Although channelrhodopsin (ChR) is a widely applied light-activated ion channel, important properties such as light adaptation, photocurrent inactivation, and alteration of the ion selectivity during continuous illumination are not well understood from a molecular perspective. Herein, we address these open questions using single-turnover electrophysiology, time-resolved step-scan FTIR, and Raman spectroscopy of fully dark-adapted ChR2. This yields a unifying parallel photocycle model integrating now all so far controversial discussed data. In dark-adapted ChR2, the protonated retinal Schiff base chromophore (RSBH+) adopts an all-trans,C=N-anti conformation only. Upon light activation, a branching reaction into either a 13-cis,C=N-anti or a 13-cis,C=N-syn retinal conformation occurs. The anti-cycle features sequential H+ and Na+ conductance in a late M-like state and an N-like open-channel state. In contrast, the 13-cis,C=N-syn isomer represents a second closed-channel state identical to the long-lived P480 state, which has been previously assigned to a late intermediate in a single-photocycle model. Light excitation of P480 induces a parallel syn-photocycle with an open-channel state of small conductance and high proton selectivity. E90 becomes deprotonated in P480 and stays deprotonated in the C=N-syn cycle. Deprotonation of E90 and successive pore hydration are crucial for late proton conductance following light adaptation. Parallel anti- and syn-photocycles now explain inactivation and ion selectivity changes of ChR2 during continuous illumination, fostering the future rational design of optogenetic tools.

scholarly journals A unifying photocycle model for light adaptation and temporal evolution of cation conductance in Channelrhodopsin-2

2018 ◽  
Author(s):  
Jens Kuhne ◽  
Johannes Vierock ◽  
Stefan Alexander Tennigkeit ◽  
Max-Aylmer Dreier ◽  
Jonas Wietek ◽  
...  
Keyword(s):  
Ion Channel ◽  
Rational Design ◽  
Open Channel ◽  
Light Adaptation ◽  
Ion Selectivity ◽  
Continuous Illumination ◽  
Channel State ◽  
Time Resolved ◽  
Open Questions ◽  
Cation Conductance

AbstractAlthough Channelrhodopsin (ChR) is a widely applied light-activated ion channel, important properties such as light-adaptation, photocurrent inactivation, and alteration of the ion selectivity during continuous illumination are not well-understood from a molecular perspective. Herein, we address these open questions using single turn-over electrophysiology, time-resolved step-scan FTIR and Raman spectroscopy of fully dark adapted ChR2. This yields a unifying parallel photocycle model explaining all data: in dark-adapted ChR2, the protonated Schiff base retinal chromophore (RSBH+) adopts an all-trans,C=N-anti conformation only. Upon light activation, a branching reaction into either a 13-cis,C=N-anti or a 13-cis,C=N-syn retinal conformation occurs. The anti-cycle features sequential H+ and Na+ conductance in a late M-like state and an N-like open-channel state. In contrast, the 13-cis,C=N-syn isomer represents a second closed-channel state identical to the long lived P480-state, which has been previously assigned to a late intermediate in a single photocycle model. Light excitation of P480 induces a parallel syn-photocycle with an open channel state of small conductance and high proton selectivity. E90 becomes deprotonated in P480 and stays deprotonated in the C=N-syn-cycle and we show that deprotonation of E90 and successive pore hydration are crucial for late proton conductance following light-adaptation. Parallel anti- and syn-photocycles explain inactivation and ion selectivity changes of ChR2 during continuous illumination, fostering the future rational design of optogenetic tools.Significance statementUnderstanding the mechanisms of photoactivated biological processes facilitates the development of new molecular tools, engineered for specific optogenetic applications, allowing the control of neuronal activity with light. Here, we use a variety of experimental and theoretical techniques to examine the precise nature of the light-activated ion channel in one of the most important molecular species used in optogenetics, channelrhodopsin-2. Existing models for the photochemical and photophysical pathway after light absorption by the molecule fail to explain many aspects of its observed behavior including the inactivation of the photocurrent under continuous illumination. We resolve this by proposing a new branched photocycle explaining electrical and photochemical channel properties and establishing the structure of intermediates during channel turnover.

scholarly journals Light and pH-induced Changes in Structure and Accessibility of Transmembrane Helix B and Its Immediate Environment in Channelrhodopsin-2

2016 ◽  
Vol 291 (33) ◽  
pp. 17382-17393 ◽  
Author(s):  
Pierre Volz ◽  
Nils Krause ◽  
Jens Balke ◽  
Constantin Schneider ◽  
Maria Walter ◽  
...  
Keyword(s):  
Open Channel ◽  
Solvent Accessibility ◽  
Transmembrane Helix ◽  
Structural Heterogeneity ◽  
Time Resolved Fluorescence ◽  
Channel State ◽  
Time Resolved ◽  
Cytoplasmic Surface ◽  
Large Conformational Change ◽  
Ph Dependent

A variant of the cation channel channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2) was selectively labeled at position Cys-79 at the end of the first cytoplasmic loop and the beginning of transmembrane helix B with the fluorescent dye fluorescein (acetamidofluorescein). We utilized (i) time-resolved fluorescence anisotropy experiments to monitor the structural dynamics at the cytoplasmic surface close to the inner gate in the dark and after illumination in the open channel state and (ii) time-resolved fluorescence quenching experiments to observe the solvent accessibility of helix B at pH 6.0 and 7.4. The light-induced increase in final anisotropy for acetamidofluorescein bound to the channel variant with a prolonged conducting state clearly shows that the formation of the open channel state is associated with a large conformational change at the cytoplasmic surface, consistent with an outward tilt of helix B. Furthermore, results from solute accessibility studies of the cytoplasmic end of helix B suggest a pH-dependent structural heterogeneity that appears below pH 7. At pH 7.4 conformational homogeneity was observed, whereas at pH 6.0 two protein fractions exist, including one in which residue 79 is buried. This inaccessible fraction amounts to 66% in nanodiscs and 82% in micelles. Knowledge about pH-dependent structural heterogeneity may be important for CrChR2 applications in optogenetics.

Molecular Design of Peptide-Fc Fusion Drugs

2019 ◽  
Vol 20 (3) ◽  
pp. 203-208 ◽  
Author(s):  
Lin Ning ◽  
Bifang He ◽  
Peng Zhou ◽  
Ratmir Derda ◽  
Jian Huang
Keyword(s):  
Rational Design ◽  
Molecular Design ◽  
Computational Design ◽  
Fusion Technology ◽  
Computational Tools ◽  
Open Questions ◽  
Biological Therapeutics ◽  
Key Steps ◽  
Computational Resources ◽  
Tools And Methods

Background:Peptide-Fc fusion drugs, also known as peptibodies, are a category of biological therapeutics in which the Fc region of an antibody is genetically fused to a peptide of interest. However, to develop such kind of drugs is laborious and expensive. Rational design is urgently needed.Methods:We summarized the key steps in peptide-Fc fusion technology and stressed the main computational resources, tools, and methods that had been used in the rational design of peptide-Fc fusion drugs. We also raised open questions about the computer-aided molecular design of peptide-Fc.Results:The design of peptibody consists of four steps. First, identify peptide leads from native ligands, biopanning, and computational design or prediction. Second, select the proper Fc region from different classes or subclasses of immunoglobulin. Third, fuse the peptide leads and Fc together properly. At last, evaluate the immunogenicity of the constructs. At each step, there are quite a few useful resources and computational tools.Conclusion:Reviewing the molecular design of peptibody will certainly help make the transition from peptide leads to drugs on the market quicker and cheaper.

scholarly journals Real-time observation of tetrapyrrole binding to an engineered bacterial phytochrome

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Yusaku Hontani ◽  
Mikhail Baloban ◽  
Francisco Velazquez Escobar ◽  
Swetta A. Jansen ◽  
Daria M. Shcherbakova ◽  
...  
Keyword(s):  
Real Time ◽  
Rational Design ◽  
Near Infrared ◽  
Fluorescent Proteins ◽  
Covalent Bond ◽  
Binding Pocket ◽  
Tissue Imaging ◽  
Covalent Attachment ◽  
Time Resolved

AbstractNear-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes are widely used for structural and functional deep-tissue imaging in vivo. To fluoresce, NIR FPs covalently bind a chromophore, such as biliverdin IXa tetrapyrrole. The efficiency of biliverdin binding directly affects the fluorescence properties, rendering understanding of its molecular mechanism of major importance. miRFP proteins constitute a family of bright monomeric NIR FPs that comprise a Per-ARNT-Sim (PAS) and cGMP-specific phosphodiesterases - Adenylyl cyclases - FhlA (GAF) domain. Here, we structurally analyze biliverdin binding to miRFPs in real time using time-resolved stimulated Raman spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations. Biliverdin undergoes isomerization, localization to its binding pocket, and pyrrolenine nitrogen protonation in <1 min, followed by hydrogen bond rearrangement in ~2 min. The covalent attachment to a cysteine in the GAF domain was detected in 4.3 min and 19 min in miRFP670 and its C20A mutant, respectively. In miRFP670, a second C–S covalent bond formation to a cysteine in the PAS domain occurred in 14 min, providing a rigid tetrapyrrole structure with high brightness. Our findings provide insights for the rational design of NIR FPs and a novel method to assess cofactor binding to light-sensitive proteins.

scholarly journals Picosecond time-resolved photon antibunching measures nanoscale exciton motion and the true number of chromophores

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Gordon J. Hedley ◽  
Tim Schröder ◽  
Florian Steiner ◽  
Theresa Eder ◽  
Felix J. Hofmann ◽  
...  
Keyword(s):  
Rational Design ◽  
Three Dimensional ◽  
Dna Origami ◽  
Fluorescent Nanoparticles ◽  
Time Resolved ◽  
Exciton Diffusion ◽  
Polymer Aggregates ◽  
True Number ◽  
Particle Level ◽  
Photon Antibunching

AbstractThe particle-like nature of light becomes evident in the photon statistics of fluorescence from single quantum systems as photon antibunching. In multichromophoric systems, exciton diffusion and subsequent annihilation occurs. These processes also yield photon antibunching but cannot be interpreted reliably. Here we develop picosecond time-resolved antibunching to identify and decode such processes. We use this method to measure the true number of chromophores on well-defined multichromophoric DNA-origami structures, and precisely determine the distance-dependent rates of annihilation between excitons. Further, this allows us to measure exciton diffusion in mesoscopic H- and J-type conjugated-polymer aggregates. We distinguish between one-dimensional intra-chain and three-dimensional inter-chain exciton diffusion at different times after excitation and determine the disorder-dependent diffusion lengths. Our method provides a powerful lens through which excitons can be studied at the single-particle level, enabling the rational design of improved excitonic probes such as ultra-bright fluorescent nanoparticles and materials for optoelectronic devices.

scholarly journals Two - Color Mid-Infrared Spectroscopy of Isoelectronic Centers in Silicon

2003 ◽  
Vol 770 ◽  
Author(s):  
N.Q. Vinh ◽  
T. Gregorkiewicz
Keyword(s):  
Excited States ◽  
Free Electron ◽  
Free Electron Laser ◽  
Materials Science ◽  
Time Resolved ◽  
Mid Infrared ◽  
Semiconductor Physics ◽  
Open Questions ◽  
Time Resolved Photoluminescence ◽  
Tunable Source

AbstractOne of the open questions in semiconductor physics is the origin of the small splittings of the excited states of bound excitons in silicon. A free electron laser as a tunable source of the mid-infrared radiation (MIR) can be used to investigate such splittings of the excited states of optical centers created by transition metal dopants in silicon. In the current study, the photoluminescence from silver and copper doped silicon is investigated by two color spectroscopy in the visible and the MIR. It is shown the PL due recombination of exciton bound to Ag and Cu is quenched upon application of the MIR beam. The time-resolved photoluminescence measurements and the quenching effects of these bands are presented. By scanning the wavelength of the free-electron laser ionization spectra of relevant traps involved in photoluminescence are obtained. The formation and dissociation of the bound excitons, and the small splittings of the effective-mass excited states are discussed. The applied experimental method allows correlation of DLTS data on trapping centers to specific channels of radiative recombination. It can be applied for spectroscopic analysis in materials science of semicondutors.

scholarly journals Down-regulation of TORC2-Ypk1 signaling promotes MAPK-independent survival under hyperosmotic stress

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Alexander Muir ◽  
Françoise M Roelants ◽  
Garrett Timmons ◽  
Kristin L Leskoske ◽  
Jeremy Thorner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Protein Kinase ◽  
Molecular Basis ◽  
Open Channel ◽  
Hyperosmotic Stress ◽  
Channel State ◽  
Dependent Protein Kinase ◽  
Hyperosmotic Shock ◽  
Dependent Protein ◽  
Intracellular Glycerol

In eukaryotes, exposure to hypertonic conditions activates a MAPK (Hog1 in Saccharomyces cerevisiae and ortholog p38 in human cells). In yeast, intracellular glycerol accumulates to counterbalance the high external osmolarity. To prevent glycerol efflux, Hog1 action impedes the function of the aquaglyceroporin Fps1, in part, by displacing channel co-activators (Rgc1/2). However, Fps1 closes upon hyperosmotic shock even in hog1∆ cells, indicating another mechanism to prevent Fps1-mediated glycerol efflux. In our prior proteome-wide screen, Fps1 was identified as a target of TORC2-dependent protein kinase Ypk1 (<xref ref-type="bibr" rid="bib30">Muir et al., 2014</xref>). We show here that Fps1 is an authentic Ypk1 substrate and that the open channel state of Fps1 requires phosphorylation by Ypk1. Moreover, hyperosmotic conditions block TORC2-dependent Ypk1-mediated Fps1 phosphorylation, causing channel closure, glycerol accumulation, and enhanced survival under hyperosmotic stress. These events are all Hog1-independent. Our findings define the underlying molecular basis of a new mechanism for responding to hypertonic conditions.

Self-Inhibition in Ca2+-Evoked Taste Responses: A Novel Tool for Functional Dissection of Salt Taste Transduction Mechanisms

1998 ◽  
Vol 79 (2) ◽  
pp. 911-921 ◽  
Author(s):  
Mamoun A. Kloub ◽  
Gerard L. Heck ◽  
John A. Desimone
Keyword(s):  
Tight Junctions ◽  
Ion Selectivity ◽  
Critical Concentration ◽  
Dependent Manner ◽  
High Concentration ◽  
Salt Taste ◽  
Cation Conductance ◽  
Taste Transduction ◽  
Transduction Mechanisms ◽  
Paracellular Pathways

Kloub, Mamoun A., Gerard L. Heck, and John A. DeSimone. Self-inhibition in Ca2+-evoked taste receptors: a novel tool for functional dissection of salt taste transduction mechanisms. J. Neurophysiol. 79: 911–921, 1998. Rat chorda tympani (CT) responses to CaCl2 were obtained during simultaneous current and voltage clamping of the lingual receptive field. Unlike most other salts, CaCl2 induced negatively directed transepithelial potentials and gave CT responses that were auto-inhibitory beyond a critical concentration. CT responses increased in a dose-dependent manner to ∼0.3 M, whereafter they decreased with increasing concentration. At concentrations where Ca2+ was self-inhibitory, it also inhibited responses to NaCl, KCl, and NH4Cl present in mixtures with CaCl2. Ca2+ completely blocked the amiloride-insensitive component of the NaCl CT response, the entire KCl-evoked CT response, and the high-concentration-domain CT responses of NH4Cl (≥0.3 M). The overlapping Ca2+-sensitivity between the responses of the three Cl− salts (Na+, K+, and NH+ 4) suggests a common, Ca2+-sensitive, transduction pathway. Extracellular Ca2+ has been shown to modulate the paracellular pathways in different epithelial cell lines by decreasing the water permeability and cation conductance of tight junctions. Ca2+-induced modulation of tight junctions is associated with Ca2+ binding to fixed negative sites. This results in a conversion of ion selectivity from cationic to anionic, which we also observed in our system through simultaneous monitoring of the transepithelial potential during CT recording. The data indicate the paracellular pathway as the stimulatory and modulatory site of CaCl2 taste responses. In addition, they indicate that important transduction sites for NaCl, KCl, and NH4Cl taste reception are accessible only through the paracellular pathways. More generally, they show that modulation of paracellular transport by Ca2+ in an intact epithelium has functional consequences at a systemic level.

Solvent exchange in a metal–organic framework single crystal monitored by dynamicin situX-ray diffraction

2017 ◽  
Vol 73 (4) ◽  
pp. 669-674 ◽  
Author(s):  
Jordan M. Cox ◽  
Ian M. Walton ◽  
Gage Bateman ◽  
Cassidy A. Benson ◽  
Travis Mitchell ◽  
...  
Keyword(s):  
Rational Design ◽  
Metal Organic Framework ◽  
X Ray Diffraction ◽  
Guest Molecules ◽  
Time Resolved ◽  
Ethanol Solvate ◽  
Significant Step ◽  
Metal Organic ◽  
Flexible Metal ◽  
Organic Framework

Understanding the processes by which porous solid-state materials adsorb and release guest molecules would represent a significant step towards developing rational design principles for functional porous materials. To elucidate the process of liquid exchange in these materials, dynamicin situX-ray diffraction techniques have been developed which utilize liquid-phase chemical stimuli. Using these time-resolved diffraction techniques, the ethanol solvation process in a flexible metal–organic framework [Co(AIP)(bpy)0.5(H2O)]·2H2O was examined. The measurements provide important insight into the nature of the chemical transformation in this system including the presence of a previously unreported neat ethanol solvate structure.

scholarly journals Evolution of Turbulent Horseshoe Vortex System in Front of a Vertical Circular Cylinder in Open Channel

Water ◽  
2019 ◽  
Vol 11 (10) ◽  
pp. 2079 ◽  
Author(s):  
Chen ◽  
Yang ◽  
Wu
Keyword(s):  
Open Channel ◽  
Evolutionary Process ◽  
Reynolds Numbers ◽  
Horseshoe Vortex ◽  
Local Scour ◽  
Time Resolved ◽  
Vortex Interactions ◽  
Initial Stage ◽  
Image Velocimetry ◽  
Underlying Mechanisms

A turbulent horseshoe vortex (HV) system around a wall-mounted cylinder in open channel is characterized by random variations in vortex features and an abundance of vortex interactions. The turbulent HV system is responsible for initiating the local scour process in front of the cylinder. The evolution of the turbulent HV system is investigated statistically and quantitatively with time-resolved particle image velocimetry. The cylinder Reynolds numbers of the flow are 8600, 10,200, and 13,600, respectively. A novel vortex tracking method was proposed to obtain the variations in position, size, and strength of the primary HV (PHV) which dominates the system most of the time. Relationships between the various features of the PHV during its evolutionary process were obtained through correlation analyses. Results show that the dimensionless mean lifespan of the PHV is about 5.0. Statistically, the downstream movement of the PHV toward the cylinder is accompanied with its bed-approaching movement and decreasing in size, and the opposite is true. The circulation strength of the PHV decreases and increases dramatically in the region downstream of its time-averaged position when the PHV approaches and departs from the cylinder, respectively. Meanwhile, mechanisms responsible for the generation, movement, variation, and disappearance of the PHV are re-investigated and enriched based on its interactions with vortices in the separation region and structures in the incoming flow. The obtained change trends of the features of the PHV and the underlying mechanisms for its evolution are valuable for predicting and controlling the initial stage of the local scour in front of cylinders.

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