Helical Contributions Mediate Light-Activated Conformational Change in the LOV2 Domain of <i>Avena sativa</i> Phototropin 1

Algae, plants, bacteria, and fungi contain flavin-binding light-oxygen-voltage (LOV) domains that function as blue light sensors to control cellular responses to light. In the second LOV domain of phototropins, called LOV2 domains, blue light illumination leads to covalent bond formation between protein and flavin that induces the dissociation and unfolding of a C-terminally attached α helix (Jα) and the N-terminal helix (A′α). To date, the majority of studies on these domains have focused on versions that contain truncations in the termini, which creates difficulties when extrapolating to the much larger proteins that contain these domains. Here, we study the influence of deletions and extensions of the A′α helix of the LOV2 domain of Avena sativa phototropin 1 (AsLOV2) on the light-triggered structural response of the protein by Fourier-transform infrared difference spectroscopy. Deletion of the A′α helix abolishes the light-induced unfolding of Jα, whereas extensions of the A′α helix lead to an attenuated structural change of Jα. These results are different from shorter constructs, indicating that the conformational changes in full-length phototropin LOV domains might not be as large as previously assumed, and that the well-characterized full unfolding of the Jα helix in AsLOV2 with short A′α helices may be considered a truncation artifact. It also suggests that the N- and C-terminal helices of phot-LOV2 domains are necessary for allosteric regulation of the phototropin kinase domain and may provide a basis for signal integration of LOV1 and LOV2 domains in phototropins

Ultrafast mid-infrared spectroscopy by chirped pulse upconversion in 1800-1000cm(-1) region

Broadband femtosecond mid-infrared pulses can be converted into the visible spectral region by chirped pulse upconversion. We report here the upconversion of pump probe transient signals in the frequency region below 1800c

Reaction dynamics of the chimeric channelrhodopsin C1C2

Channelrhodopsin (ChR) is a key protein of the optogenetic toolkit. C1C2, a functional chimeric protein of Chlamydomonas reinhardtii ChR1 and ChR2, is the only ChR whose crystal structure has been solved, and thus uniquely suitable for structure-based analysis. We report C1C2 photoreaction dynamics with ultrafast transient absorption and multi-pulse spectroscopy combined with target analysis and structure-based hybrid quantum mechanics/molecular mechanics calculations. Two relaxation pathways exist on the excited (S-1) state through two conical intersections Cl-1 and Cl-2, that are reached via clockwise and counter-clockwise rotations: (i) the C13=C14 isomerization path with 450 fs via Cl-1 and (ii) a relaxation path to the initial ground state with 2.0 ps and 11 ps via Cl-2, depending on the hydrogen-bonding network, hence indicating active-site structural heterogeneity. The presence of the additional conical intersection Cl-2 rationalizes the relatively low quantum yield of photoisomerization (30 +/- 3%), reported here. Furthermore, we show the photoreaction dynamics from picoseconds to seconds, characterizing the complete photocycle of C1C2

Photochemie und Signaltransduktion von Blaulichtrezeptorproteinen aus photosynthetisierenden Mikroorganismen

Die lichtaktivierte Kinase Phototropin aus Chlamydomonas reinhardtii, die photoaktivierte Adenylatcyclase (PAC) aus Euglena gracilis und das BLUF-Protein Slr1694 aus Synechocystis sp. PCC 6803 wurden in Hinblick auf die molekularen Details der primären photochemischen Prozesse sowie der Signalweiterleitung untersucht. Phototropin wurde mit Hilfe von Arginin aus Escherichia coli in Milligramm Mengen isoliert. Ohne Arginin wurde E. coli cAMP Rezeptorprotein assoziiert aufgefunden, welches eine hohe Homologie zu einer cAMP aktivierten Kinase aus C. reinhardtii besitzt. Volllängen Phototropin bildet wie einzelne LOV-Domänenkonstrukte ohne Kinasedomäne den Flavin-Triplettzustand und das kovalente Cysteinyl-Addukt. Der Zerfall des Signalzustandes ist in Anwesenheit von ATP beschleunigt und deutet auf Photorezeptor-Kinase Interaktion hin. Strukturelle Änderungen in der Kinasedomäne wurden durch FTIR-Differenzspektroskopie gezeigt. Über ELDOR-Spektroskopie wurde der Abstand der Photorezeptordomänen auf etwa 25 Angstrom bestimmt. Mutationen in Slr1694 an S28, N31 und W91 zeigten keine konservierten Einfluss auf die Dynamik des Signalzustands. Die Entfernung der Seitenkette von S28 führte zu einer 15 nm Rotverschiebung des Absorptionsspektrums aufgrund veränderter Wasserstoffbrückenkoordination des Kofaktors. Die Einführung von positiv geladenen Seitenketten an Stelle von N31 erhöhte die Kofaktorbindung von phosphorylierten Flavinen. Künstliche Kofaktoren wie Roseoflavin konnten in Slr1694 durch Koexpression eines prokaryotischen Flavintransporters erreicht werden. Die Rolle von M152 in PAC für die Signalweiterleitung wurde anhand der lichtaktivierten cAMP Synthese-Aktivität gezeigt. Durch ultraschnelle IR-Spektroskopie wurde die Beteiligung der Seitenketten von Y8 sowie Q50 bestätigt und eine genauere Beschreibung der Wasserstoffbrücken im langlebigen Signalzustand ermöglicht.The light activated kinase Phototropin from Chlamydomonas reinhardtii, the photoactivated adenylylcyclase (PAC) from Euglena gracilis and the BLUF protein Slr1694 from Synechocystis sp. PCC 6803 were investigated concerning the molecular details of the primary photochemistry as well as signal transduction. Phototropin was isolated from Escherichia coli in mg amounts after solubilization with arginine. Without arginine E. coli cAMP receptor protein, which shows high homology to a cAMP activated kinase from C. reinhardtii, was copurified. Full length Phototropin shows similar photochemistry to LOV-domain containing proteins without the kinase including triplet and covalent cysteinyl adduct formation. Signaling state decay is accelerated in the presence of ATP and suggests photoreceptor-kinase interaction. FTIR spectroscopy showed light induced structural changes in the kinase domain. The distance of the photoreceptor domains of 25 Angstrom was determined by ELDOR spectroscopy. Mutation of the side chains of S28, N31 and W91 in Slr1694 showed no conserved influence on the dynamic of the signaling state. Removal of the hydroxyl group of S28 lead to a 15 nm red shift of the absorption spectrum as a result of altered hydrogen bond coordination of the cofactor. Introduction of positively charged side chains at the position of N31 strengthened the binding of phosphorylated flavins. An artificial flavin like roseoflavin was introduced in Slr1694 by coexpression of a bacterial flavin transporter. The essential role of M152 in PAC for signal transduction was shown by determination of light activated cAMP synthesis activity. Ultrafast IR spectroscopy confirmed the contribution of Y8 and Q50 in the photocycle and gave a more detailed description of the hydrogen bonding situation in the signaling state

Optogenetic Tools in the Molecular Spotlight

The rise of optogenetics as a standard technique to non-invasively probe and monitor biological function created an immense interest in the molecular function of photosensory proteins. These photoreceptors are usually protein/pigment complexes that translate light into biological information and have become essential tools in cell biology and neurobiology as their function is genetically encoded and can be conveniently delivered into a given cell. Like for fluorescent proteins that quickly became invaluable as genetically encodable reporters in microscopy and imaging, variants of photosensory proteins with customized sensitivity and functionality are nowadays in high demand. In this ebook we feature reviews and original research on molecular approaches from synthetic biology and molecular spectroscopy to computational molecular modelling that all aspire to elucidate the molecular prerequisites for the photosensory function of the given proteins. The principle property of changing activity of biological function simply by application of light is not only very attractive for cell biology, it also offers unique opportunities for molecular studies as excitation can be controlled with high time precision. Especially in spectroscopy the usually fully reversible photoactivation of photosensory proteins allows researchers to to perform time resolved studies with up to femtosecond resolution. In addition, functional variants can be investigated and quickly screened in common biochemical experiments. The insights that are obtained by the here presented various yet complementary methods will ultimately allow us write the script for a molecular movie from excitation of the protein by a photon to activation of its biological function. Such deep understanding does not only provide unique insights into the dynamics of protein function, it will also ultimately enable us to rationally design novel optogenetic tools to be used in cell biology and therapy

Ultrafast Laser Energy Density and Retinal Absorption Cross-Section Determination by Saturable Absorption Measurements

Laser pulse nonlinear transmission measurements through saturable absorbers of known absorption parameters allow the measurement of their energy density. On the other hand, nonlinear transmission measurements of laser pulses of known energy density through absorbing media allow their absorption parameter determination. The peak energy density w0P of second harmonic pulses of a mode-locked titanium sapphire laser at wavelength λP = 400 nm is determined by nonlinear energy transmission measurement TE through the dye ADS084BE (1,4-bis(9-ethyl-3-car-bazovinylene)-2-methoxy-5-(2’-ethyl-hexyloxy)-benzene) in tetrahydrofuran. TE(w0P) calibration curves are calculated for laser pulse peak energy density reading w0P from measured pulse energy transmissions TE. The ground-state absorption cross-section σP and the excited-state absorption cross-section σex at λP, and the number density N0 of the retinal Schiff base isoform RetA in pH 7.4 buffer of the blue-light adapted recombinant rhodopsin fragment of the histidine kinase rhodopsin HKR1 from Chlamydomonas reinhardtii were determined by picosecond titanium sapphire second harmonic laser pulse energy transmission measurement TE through RetA as a function of laser input peak energy density w0P. The complete absorption cross-section spectrum