Atomistic Insight into the Role of Threonine 127 in the Functional Mechanism of Channelrhodopsin-2

Channelrhodopsins (ChRs) belong to the unique class of light-gated ion channels. The structure of channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2) has been resolved, but the mechanistic link between light-induced isomerization of the chromophore retinal and channel gating remains elusive. Replacements of residues C128 and D156 (DC gate) resulted in drastic effects in channel closure. T127 is localized close to the retinal Schiff base and links the DC gate to the Schiff base. The homologous residue in bacteriorhodopsin (T89) has been shown to be crucial for the visible absorption maximum and dark–light adaptation, suggesting an interaction with the retinylidene chromophore, but the replacement had little effect on photocycle kinetics and proton pumping activity. Here, we show that the T127A and T127S variants of CrChR2 leave the visible absorption maximum unaffected. We inferred from hybrid quantum mechanics/molecular mechanics (QM/MM) calculations and resonance Raman spectroscopy that the hydroxylic side chain of T127 is hydrogen-bonded to E123 and the latter is hydrogen-bonded to the retinal Schiff base. The C=N–H vibration of the Schiff base in the T127A variant was 1674 cm−1, the highest among all rhodopsins reported to date. We also found heterogeneity in the Schiff base ground state vibrational properties due to different rotamer conformations of E123. The photoreaction of T127A is characterized by a long-lived P2380 state during which the Schiff base is deprotonated. The conservative replacement of T127S hardly affected the photocycle kinetics. Thus, we inferred that the hydroxyl group at position 127 is part of the proton transfer pathway from D156 to the Schiff base during rise of the P3530 intermediate. This finding provides molecular reasons for the evolutionary conservation of the chemically homologous residues threonine, serine, and cysteine at this position in all channelrhodopsins known so far

Temporal evolution of helix hydration in a light-gated ion channel correlates with ion conductance

The discovery of channelrhodopsins introduced a new class of light-gated ion channels, which when genetically encoded in host cells resulted in the development of optogenetics. Channelrhodopsin-2 from Chlamydomonas reinhardtii, CrChR2, is the most widely used optogenetic tool in neuroscience. To explore the connection between the gating mechanism and the influx and efflux of water molecules in CrChR2, we have integrated light-induced time- resolved infrared spectroscopy and electrophysiology. Cross-correlation analysis revealed that ion conductance tallies with peptide backbone amide I vibrational changes at 1,665(−) and 1,648(+) cm−1. These two bands report on the hydration of transmembrane α-helices as concluded from vibrational coupling experiments. Lifetime distribution analysis shows that water influx proceeded in two temporally separated steps with time constants of 10 μs (30%) and 200 μs (70%), the latter phase concurrent with the start of ion conductance. Water efflux and the cessation of the ion conductance are synchronized as well, with a time constant of 10 ms. The temporal correlation between ion conductance and hydration of helices holds for fast (E123T) and slow (D156E) variants of CrChR2, strengthening its functional significance

Personalized web learning: merging Open Educational Resources into adaptive courses for higher education

In this paper, educational and technical challenges for applying learning pathways in Massive(ly) Open Online Courses in higher education are outlined. The authors argue that quality issues and didactical concerns may be overcome by (1) reverting to small Open Educational Resources that are (2) adaptively joined into concise courses by considering (3) predefined learning pathways with proper semantic annotations and (4) the observation of learner behaviour. Such a merger does not only require conceptual work and corresponding support tools, but also a new meta data format and an engine which interprets the semantic annotations as well as the measures of learner’s actions. These factors are then turned into didactically meaningful recommendations for the next learning steps, thereby creating a personalized learning pathway for each learner. The EU FP7 project INTUITEL is introduced, which has already contributed to the conceptual work and is currently developing the software to achieve these tasks. (DIPF/Orig.

Learning Pathway Recommendation based on a Pedagogical Ontology and its Implementation in Moodle

When learners may select among different alternatives, or are guided to do so by an adaptive learning environment (ALE), it is generally meaningful to discuss the concept of different learning pathways. Pedagogically, these learning pathways may either be defined macroscopically, e.g. in terms of desired learning outcomes or competencies, or microscopically in terms of a didactical model for individual knowledge objects. In this contribution we consider such learning pathways from a pedagogical point of view and then establish a mathematical model for their traversal by a learner and for the analysis of his behavior. This model is implemented in a novel ALE provided by the EU FP7 project INTUITEL, introduced in its Moodle version as concrete example

A SMS with a KISS. Towards a pedagogical design of a metadata system for adaptive learning pathways

While there are many ontologies and metadata standards available in the field of Technology Enhanced Learning that support adaptive learning pathways, hardly any of these ontologies and metadata standards are ready for use and applied in professional education. Thus we suggest a simple metadata system (SMS) that considers educational theories, is kept simple and stupid (KISS) to increase usability and delivers pedagogical benefits

Personalized web learning: merging Open Educational Resources into adaptive courses for higher education

In this paper, educational and technical challenges for applying learning pathways in Massive(ly) Open Online Courses in higher education are outlined. The authors argue that quality issues and didactical concerns may be overcome by (1) reverting to small Open Educational Resources that are (2) adaptively joined into concise courses by considering (3) predefined learning pathways with proper semantic annotations and (4) the observation of learner behaviour. Such a merger does not only require conceptual work and corresponding support tools, but also a new meta data format and an engine which interprets the semantic annotations as well as the measures of learner’s actions. These factors are then turned into didactically meaningful recommendations for the next learning steps, thereby creating a personalized learning pathway for each learner. The EU FP7 project INTUITEL is introduced, which has already contributed to the conceptual work and is currently developing the software to achieve these tasks. (DIPF/Orig.

INTUITEL - Intelligent Tutorial Interface for Technology Enhanced Learning

The EU FP7 project INTUITEL provides a novel adaptive learningenvironment, which is neither test-driven nor curriculum-based. Although based on ontologies, it extends these by an innovative concept of learning pathwaysand a subsequent ranking algorithm. The resulting environment is in line with pedagogical models and cognitive theories and will be integrated in five differentmarket leading learning platforms. (DIPF/Orig.

Indication for a Radical Intermediate Preceding the Signaling State in the LOV Domain Photocycle

Bauer C, Rabl C-R, Heberle J, Kottke T. Indication for a Radical Intermediate Preceding the Signaling State in the LOV Domain Photocycle. PHOTOCHEMISTRY AND PHOTOBIOLOGY. 2011;87(3):548-553

The retinal structure of channelrhodopsin-2 assessed by resonance Raman spectroscopy

Nack M, Radu I, Bamann C, Bamberg E, Heberle J. The retinal structure of channelrhodopsin-2 assessed by resonance Raman spectroscopy. FEBS Letters. 2009;583(22):3676-3680.Channelrhodopsin-2 mediates phototaxis in green algae by acting as a light-gated cation channel. As a result of this property, it is used as a novel optogenetic tool in neurophysiological applications. Structural information is still scant and we present here the first resonance Raman spectra of channelrhodopsin-2. Spectra of detergent solubilized and lipid-reconstituted protein were recorded under pre-resonant conditions to exclusively probe retinal in its electronic ground state. All-trans retinal was identified to be the favoured configuration of the chromophore but significant contributions of 13-cis were detected. Pre-illumination hardly changed the isomeric composition but small amounts of presumably 9-cis retinal were found in the light-adapted state. Spectral analysis suggested that the Schiff base proton is strongly hydrogen-bonded to a nearby water molecule. (C) 2009 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved