Chemical Magnetoreception: Bird Cryptochrome 1a Is Excited by Blue Light and Forms Long-Lived Radical-Pairs

Cryptochromes (Cry) have been suggested to form the basis of light-dependent magnetic compass orientation in birds. However, to function as magnetic compass sensors, the cryptochromes of migratory birds must possess a number of key biophysical characteristics. Most importantly, absorption of blue light must produce radical pairs with lifetimes longer than about a microsecond. Cryptochrome 1a (gwCry1a) and the photolyase-homology-region of Cry1 (gwCry1-PHR) from the migratory garden warbler were recombinantly expressed and purified from a baculovirus/Sf9 cell expression system. Transient absorption measurements show that these flavoproteins are indeed excited by light in the blue spectral range leading to the formation of radicals with millisecond lifetimes. These biophysical characteristics suggest that gwCry1a is ideally suited as a primary light-mediated, radical-pair-based magnetic compass receptor

Weak temperature dependence of P (+) H A (-) recombination in mutant Rhodobacter sphaeroides reaction centers

International audienceIn contrast with findings on the wild-type Rhodobacter sphaeroides reaction center, biexponential P (+) H A (-) → PH A charge recombination is shown to be weakly dependent on temperature between 78 and 298 K in three variants with single amino acids exchanged in the vicinity of primary electron acceptors. These mutated reaction centers have diverse overall kinetics of charge recombination, spanning an average lifetime from ~2 to ~20 ns. Despite these differences a protein relaxation model applied previously to wild-type reaction centers was successfully used to relate the observed kinetics to the temporal evolution of the free energy level of the state P (+) H A (-) relative to P (+) B A (-) . We conclude that the observed variety in the kinetics of charge recombination, together with their weak temperature dependence, is caused by a combination of factors that are each affected to a different extent by the point mutations in a particular mutant complex. These are as follows: (1) the initial free energy gap between the states P (+) B A (-) and P (+) H A (-) , (2) the intrinsic rate of P (+) B A (-) → PB A charge recombination, and (3) the rate of protein relaxation in response to the appearance of the charge separated states. In the case of a mutant which displays rapid P (+) H A (-) recombination (ELL), most of this recombination occurs in an unrelaxed protein in which P (+) B A (-) and P (+) H A (-) are almost isoenergetic. In contrast, in a mutant in which P (+) H A (-) recombination is relatively slow (GML), most of the recombination occurs in a relaxed protein in which P (+) H A (-) is much lower in energy than P (+) H A (-) . The weak temperature dependence in the ELL reaction center and a YLH mutant was modeled in two ways: (1) by assuming that the initial P (+) B A (-) and P (+) H A (-) states in an unrelaxed protein are isoenergetic, whereas the final free energy gap between these states following the protein relaxation is large (~250 meV or more), independent of temperature and (2) by assuming that the initial and final free energy gaps between P (+) B A (-) and P (+) H A (-) are moderate and temperature dependent. In the case of the GML mutant, it was concluded that the free energy gap between P (+) B A (-) and P (+) H A (-) is large at all times

Frequently asked questions about chlorophyll fluorescence, the sequel

[EN] Using chlorophyll (Chl) a fluorescence many aspects of the photosynthetic apparatus can be studied, both in vitro and, noninvasively, in vivo. Complementary techniques can help to interpret changes in the Chl a fluorescence kinetics. Kalaji et al. (Photosynth Res 122: 121-158, 2014a) addressed several questions about instruments, methods and applications based on Chl a fluorescence. Here, additionalChl a fluorescence-related topics are discussed again in a question and answer format. Examples are the effect of connectivity on photochemical quenching, the correction of F-V/F-M values for PSI fluorescence, the energy partitioning concept, the interpretation of the complementary area, probing the donor side of PSII, the assignment of bands of 77 K fluorescence emission spectra to fluorescence emitters, the relationship between prompt and delayed fluorescence, potential problems when sampling tree canopies, the use of fluorescence parameters in QTL studies, the use of Chl a fluorescence in biosensor applications and the application of neural network approaches for the analysis of fluorescence measurements. The answers draw on knowledge fromdifferent Chl a fluorescence analysis domains, yielding in several cases new insights.Kalaji, H.; Schansker, G.; Brestic, M.; Bussotti, F.; Calatayud, A.; Ferroni, L.; Goltsev, V.... (2017). Frequently asked questions about chlorophyll fluorescence, the sequel. 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Measuring and Modeling Excitation Dynamics in Photosystem I

0\. Titel 1\. Einleitung 6 2\. Experimentelle Methoden 33 3\. Messungen 51 4\. Modellierungen 79 5\. Dynamik angeregter Zustände in Photosystem I 121 6\. Zusammenfassung 142 7\. Literatur 146In der vorliegenden Arbeit wird die Dynamik von Transfer und Abbau der Anregungsenergie in einem cyanobakteriellen PS I Kernantenne-RZ-Komplex aus Synechococcus el. untersucht durch Kombination von Fluoreszenzinduktion mit ps-zeitaufgelösten Fluoreszenzmessungen bei Temperaturen zwischen 5 K und 300 K unter besonderer Beachtung des Redoxzustandes des primären Donators P700. Das gemessene Fluoreszenzverhalten wird durch Simulation der Anregungsenergiedynamik auf der Grundlage struktureller and spektraler Daten modelliert. Erstmalig wurde ein Unterschied in der Löscheffizienz des PS I Kernantenne-RZ- Komplexes mit offenem bzw. geschlossenem RZ sowohl durch Fluoreszenzinduktion und ps- Fluoreszenz als auch durch quasistationäre Spektroskopie aufgelöst. Der Unterschied in der Fluoreszenzausbeute von 12± 5 % ist nicht auf spektrale Unterschiede zwischen offenem und geschlossenem System zurückzuführen, sondern auf eine ~3 ps Differenz in der Lebensdauer des Anregungszustandes. Damit ist im geschlossenen PS I die Löschung etwas langsamer als im offenen. Mittels zeitaufgelöster Fluoreszenz wurden nicht-konservative Transferspektren sowohl für ZT als auch für 5 K gefunden, folglich ist der gelöschte Zustand nicht völlig thermisch equilibriert, was eine Trap- Limitierung der Anregungsdynamik ausschließt. Aus der Lebensdauer der Anregung bei 5 K in geschlossenem PS I kann ein A720-P700 Abstand von ~3,5 nm abgeschätzt werden. Der beobachtete Austausch von Anregungen zwischen unterschiedlichen Monomeren kann durch Lokalisierung einiger A720 Pigmente in der Verbindungsdomäne des Trimers erklärt werden. Struktur/spektral gestützte Simulation der Anregungsdynamik erweist die Notwendigkeit, einige A708 Pigmente unmittelbar am RZ zu plazieren, um die transiente Population von P700 zu erhöhen. Auf der Grundlage dieser Anordnung der roten Pigmente ergibt die kinetische Modellierung optimale Parameterwerte von (0.5 ps)-1 intrinsischer Ladungstrennungsrate und 7,3 nm Försterradius für die Reproduktion der gemessenen Fluoreszenz. Weiterhin zeigt die Simulation eine Störung des thermischen Gleichgewichts im roten Spektralbereich und die entscheidende Rolle der roten Pigmente. Die Dynamik von geschlossenem PS I kann durch schnelle Löschung mittels interner Konversion beschrieben werden, wenn das nicht-oxidierte P700 Chl maximal bei 685 nm absorbiert. Ein Test des Einflusses der verschiedenen Parameter zeigt, daß für offenes PS I der Anregungszerfall durch ausgewogene Kinetik statt limitierender Fälle beschrieben wird.In this work, dynamics of excitation energy transfer and decay in a cyanobacterial PS I core antenna-RC-complex from Synechococcus el. is studied combining fluorescence induction techniques with picosecond fluorescence lifetime measurements at temperatures between 5 K and 300 K with special attention to the oxidation state of the primary donor P700. The measured fluorescence behaviour is modeled by simulations of the excited state transfer and decay based on structral and spectral information. For the first time, a difference in the quenching efficiency of the PS I core- antenna-RC-complex with open vs. closed RC was resolved both by fluorescence induction and ps- fluorescence as well as by quasi steady state spectroscopy. As the form of the steady state spectra at RT is (nearly) identical, the found difference of 12± 5 % in fluorescence yield is not due to spectral differences between the open and closed RC states but to a ~3 ps difference in the overall excitation decay lifetime. Thus, in closed PS I the excited state is quenched slightly worse than in open PS I. Time-resolved fluorescence could resolve non-conservative transfer spectra both at RT and at 5 K, indicating that the quenched state of the complex is not fully equilibrated. As a consequence, trap limitation of the excited state kinetics can be excluded. From the excited state lifetime at 5 K in closed PS I complexes a A720-P700 distance of ~3.5 nm is calculated. The observed inter-monomer exchange of excitation energy suggests the localization of some A720 pigments in the connecting domain of the trimeric unit. Structural/spectral information based simulation of the excited state dynamics reveales the necessiety for some A708 pigments to be placed next to the RC domain to increase the transient population of P700. The kinetics observed on a model with such arrangement of the red pigments yields optimal parameters as an intrinsic charge separation rate constant of (0.5 ps)-1 and a Förster radius of 7.3 nm for the reproduction of the measured fluorescence. Above that, the simulation shows an equilibration pertubation over the whole red wing of the emission and the determining influence of the red pigments. The kinetics in closed PS I can be described by fast quenching via internal conversion if the non-oxidized P700 Chl is set to maximal absorption at 685 nm. Testing the influence of the various parameters leads to the conclusion that for open PS I, the observable excited state decay is characterized by well-balanced kinetics rather than limiting cases

What can optical spectroscopy contribute to understanding protein dynamics ?

The short answer to the title question is: "a lot". It was transient absorption spectroscopy on geminate recombination in myoglobin that led Hans Frauenfelder to constructing his picture of protein's hierarchical energy landscape [1]. And even before that (in 1973), Joseph Lakowicz and Gregorio Weber at UIUC used quenching of tryptophan fluorescence by oxygen diffusing to solvent-inaccessible protein regions to conclude that "proteins, in general, undergo rapid structural fluctuations on the nanosecond time scale " [2]. The not-so-short answer is that the present text is written at a point where, after a decade of applying transient absorption spectroscopy to understand light induced electron transfer in a variety of enzymes, I am about to change the angle of attack and ask how these techniques and enzymes could be of help to solve some problems that are addressed in the IBS environment, namely protein dynamics, both structural and functional. It is for this reason that the answer will have to be delayed to the third and final part of this opus, "future", that deals with the perspectives. Meanwhile, the first part, "past", will be dedicated to showing on the example of the "paradigm" enzyme -DNA photolyase (the yellow egg hereunder)-, what transient absorption spectroscopy is capable of and the middle part, "present" dresses a short review into various experimental approaches currently used to obtain insight into protein dynamics. In the final section, I will delineate ways how optical spectroscopy could interact with projects existing or emerging in the protein dynamics community at IBS and thus contribute elements of an answer to the title question

Passive Experiments for Monitoring Mining Operations by Dragline at Kuzbass Open Pits – Estimation of Coal Losses

The natural conditions for the formation of coal deposits in different regions of the globe are the same, all of them belong to reservoir sedimentary deposits and differ only in the degree of metamorphism and tectonic disturbances. In this regard, coal deposits of the Kuznetsk basin (Kuzbass, Western Siberia, Russia)) that have no analogues in nature are unique. Here are all sorts of options for the occurrence of coal seams both in terms of their thickness, dip angle, number, and the degree of disturbance by plicative and disjunctive disturbances. The article presents some results of research on ways to reduce coal losses in open pit mining during its extraction by draglines. The study was carried out on the example of deposits in Kemerovo region with coal seams in an inclined and steep formations, which allows analyzing the possibilities of applying the proposed technological solutions in the widest range of specific mining and geological conditions

The CAL(AI)2DOSCOPE: a microspectrophotometer for accurate recording of correlated absorbance and fluorescence emission spectra

International audienceMicrospectrometers are (sometimes bulky) spectrometers specifically designed for the study of microscopic samples. A variety of instruments have been developed that combine spot sizes down to sub-microns with spec-tral ranges from ultraviolet (UV) to infra-red (IR) for various optical spectroscopy modalities such as absorbance, transmit-tance, fluorescence or vibrational spec-troscopies. If, on top of being tiny, the samples are delicate objects such as protein microcrystals, other challenges than just tight and achromatic focusing arise: sample optical anisotropy, large refractive index and demanding envi-ronmental requirements make most commercially available devices unsuited. Therefore, at many places where micro-spectroscopy is required (for example at synchrotron sources to investigate protein crystals), home-made microspectrome-ters have been developed in recent years that strive to meet the above-mentioned challenges.1At the European Synchrotron Radiation Facility in Grenoble, France (ESRF), a dedicated system based on three mutu-ally aligned mirror objectives has been installed (“Cryobench”)2,3 that combines the absorption (A), fluorescence (F) and Raman modalities with the possibility of rotational adjustment of the sample (via a goniometric support), and the option to cool the sample down to 100 K due to a stream of gaseous nitrogen. Our experience with the powerful possibili-ties of this system, and the realisation of its limitations prompted us to design a next generation microspectrometer dubbed “CAL(AI)2DOSCOPE” which is presented in this article. The biological samples we study (fluorescent proteins used as genetically encoded markers for advanced fluorescence microscopy) exhibit an intricate behaviour highly sensitive to their micro-environment and notably to their illumination history. As a consequence, their absorption and fluo-rescence (giving access to their photo-physical properties) cannot be studied independently, but rather need to be followed (quasi-) simultaneously, requir-ing coordination in space and time. Existing solutions such as crossed beams (e.g. ESRF Cryobench)4 or optics rear-rangement (e.g. SwissLightSource)5 are not fully compatible with these require-ments. Therefore, we developed the alternative solution of a common optical path for both A and F, coupled to rapid switching of light sources and detectors by upstream mechanical shutters