15 research outputs found
Chromophore Protonation State Controls Photoswitching of the Fluoroprotein asFP595
Fluorescent proteins have been widely used as genetically encodable fusion tags for biological imaging. Recently, a new class of fluorescent proteins was discovered that can be reversibly light-switched between a fluorescent and a non-fluorescent state. Such proteins can not only provide nanoscale resolution in far-field fluorescence optical microscopy much below the diffraction limit, but also hold promise for other nanotechnological applications, such as optical data storage. To systematically exploit the potential of such photoswitchable proteins and to enable rational improvements to their properties requires a detailed understanding of the molecular switching mechanism, which is currently unknown. Here, we have studied the photoswitching mechanism of the reversibly switchable fluoroprotein asFP595 at the atomic level by multiconfigurational ab initio (CASSCF) calculations and QM/MM excited state molecular dynamics simulations with explicit surface hopping. Our simulations explain measured quantum yields and excited state lifetimes, and also predict the structures of the hitherto unknown intermediates and of the irreversibly fluorescent state. Further, we find that the proton distribution in the active site of the asFP595 controls the photochemical conversion pathways of the chromophore in the protein matrix. Accordingly, changes in the protonation state of the chromophore and some proximal amino acids lead to different photochemical states, which all turn out to be essential for the photoswitching mechanism. These photochemical states are (i) a neutral chromophore, which can trans-cis photoisomerize, (ii) an anionic chromophore, which rapidly undergoes radiationless decay after excitation, and (iii) a putative fluorescent zwitterionic chromophore. The overall stability of the different protonation states is controlled by the isomeric state of the chromophore. We finally propose that radiation-induced decarboxylation of the glutamic acid Glu215 blocks the proton transfer pathways that enable the deactivation of the zwitterionic chromophore and thus leads to irreversible fluorescence. We have identified the tight coupling of trans-cis isomerization and proton transfers in photoswitchable proteins to be essential for their function and propose a detailed underlying mechanism, which provides a comprehensive picture that explains the available experimental data. The structural similarity between asFP595 and other fluoroproteins of interest for imaging suggests that this coupling is a quite general mechanism for photoswitchable proteins. These insights can guide the rational design and optimization of photoswitchable proteins
Molekulardynamik Simulationen Photoaktivierter Prozesse in kondensierter Phase
Understanding light-driven processes in condensed phase is a major goal of the bio- and nanosciences. The underlying molecular mechanisms in terms of the molecular dynamics are typically governed by sub-picosecond atomic motion. Such ultra-fast timescales are very challenging to probe by experiment. The present thesis aimes at characterizing by means of molecular dynamics (MD) simulations the molecular mechanisms of three photochemical processes in condensed phase, the photoswitching mechanism of the fluoroprotein asFP595, the deactivation of an excited cytosine-guanine DNA base pair, and the optical contraction of a photoswitchable polyazobenzene peptide. Our simulations provide detailed structural and dynamic information about these processes at a resolution well beyond what is achievable experimentally. By using an ab initio QM/MM excited state MD strategy together with explicit surface hopping (asFP595 and DNA) and force-probe MD (polyazobenzene peptides), it was not only possible to quantitatively explain experimental results (such as quantum yields, excited state lifetimes, and force-extension curves), but also to make predictions that are rigorously testable, and in parts have already been tested, by experimental means. The detailed understanding of the molecular mechanism is a key step towards the rational improvement and design of photoactivatable systems, as exemplarily demonstrated for a polyazobenzene peptide.Licht-getriebene Prozesse in kondensierter Phase im Detail zu verstehen ist ein Hauptziel der Bio- und Nanowissenschaften. Die molekulare Dynamik der zu Grunde liegenden Mechanismen lĂ€uft typischerweise auf der sub-Pikosekunden Zeitskala ab. Derart ultra-schnelle Atombewegungen sind experimentell nur sehr schwer zu erfassen. Das Ziel der vorliegenden Arbeit ist es, an Hand von Molekulardynamik (MD) Simulationen die molekularen Mechanismen von drei verschiedenden photochemischen Prozessen in kondensierter Phase aufzuklĂ€ren, den Photoschalt-Mechanismus des Fluoroproteins asFP595, die Deaktivierung eines angeregten Cytosin-Guanin DNA Basenpaares, und die optische Kontraktion eines photoschaltbaren Polyazobenzol Peptides. Unsere Simulationen liefern detaillierte strukturelle und dynamische Informationen ĂŒber diese Prozesse mit einer Auflösung, die jenseits der experinmentell erreichbaren Genauigkeit liegt. Die Anwendung von ab initio QM/MM MD Simulationen im lichtangeregten Zustand zusammen mit einem diabatischen surface hopping Algorithmus (asFP595 und DNA) und force-probe MD (Polyazobenzol Peptide) ermöglichte es nicht nur, experimentelle Ergebnisse quantitativ zu erklĂ€ren (Quantenausbeuten, Lebenszeiten des angeregten Zustandes, Kraft-Auslenkungs Kurven), sondern auch Vorhersagen zu treffen, die experimentell ĂŒberprĂŒfbar sind und zum Teil auch schon ĂŒberprĂŒft wurden. Das VerstĂ€ndinis des molekularen Mechanismus ist ein SchlĂŒsselschritt auf dem Weg zum rationellen Design photoschaltbarer Systeme, wie in dieser Arbeit beispielhaft fĂŒr ein Polyazobenzol Peptid demonstriert
Molecular Mechanism of a Green-Shifted, pH-Dependent Red Fluorescent Protein mKate Variant
Fluorescent proteins that can switch between distinct colors have contributed significantly to modern biomedical imaging technologies and molecular cell biology. Here we report the identification and biochemical analysis of a green-shifted red fluorescent protein variant GmKate, produced by the introduction of two mutations into mKate. Although the mutations decrease the overall brightness of the protein, GmKate is subject to pH-dependent, reversible green-to-red color conversion. At physiological pH, GmKate absorbs blue light (445 nm) and emits green fluorescence (525 nm). At pH above 9.0, GmKate absorbs 598 nm light and emits 646 nm, far-red fluorescence, similar to its sequence homolog mNeptune. Based on optical spectra and crystal structures of GmKate in its green and red states, the reversible color transition is attributed to the different protonation states of the cis-chromophore, an interpretation that was confirmed by quantum chemical calculations. Crystal structures reveal potential hydrogen bond networks around the chromophore that may facilitate the protonation switch, and indicate a molecular basis for the unusual bathochromic shift observed at high pH. This study provides mechanistic insights into the color tuning of mKate variants, which may aid the development of green-to-red color-convertible fluorescent sensors, and suggests GmKate as a prototype of genetically encoded pH sensors for biological studies
Theoretical study on the photoswitching mechanism of negative reversibly photoswitchable fluorescent proteins
105 p.Reversibly photoswitchable uorescent proteins (RSFPs) are genetically engineered proteins that can be switched by light absorption between a uorescent ON state and a dark OFF state. Among other applications they allow to increase the resolution beyond the difraction limit in cell imaging by uorescence microscopy. RSFPs have extended the possibilities of uorescence microscopy and other biotechnological tools, but the development of their properties is still far from being rationally designed. Thus, there might be much room for improvement if we manage to understand the switching mechanisms. The switching mechanisms in several negative RSFPs is being elucidated but still under debate. In this thesis I extend the theoretical knowledge about the photoswitching of negative RSFPs by studying the excited-state potential energy surface of both the ON- and the OFF-state. I compare three RSFPs, namely IrisFP, Dronpa and a fast switching single mutant of Dronpa called Dronpa2 to search for the origin of their diferent switching quantum yields in the ON-state. For the OFF-state, the results of the combined quantum mechanics{molecular mechanics (QM/MM) calculations show that chromophore photoisomerization happens in its neutral form and is followed by ground state deprotonation. This is in agreement with a very recent ultrafast absorption spectroscopy study for IrisFP, and studies on Dronpa and other negative RSFPs. Although the experimental results in both proteins show the same steps, I found that they have diferent processes at the atomic level due to structural and electrostatical diferences, but leading to the same intermediates. This is in contrast to the ON-state where I get the same picture for the three proteins studied, identifying the conical intersection that quenches the uorescence and controls the photoswitching quantum yield. The major diference between the three proteins in terms of uorescence and photoswitching characteristics comes from the diferent sterical environment produced by the residue 159, which is a methionine in diferent isomers in IrisFP and Dronpa and a smaller threonine that allows a faster isomerization of the chromophore in Dronpa2
Mécanismes de photo-commutation réversible des protéines fluorescentes
La propriĂ©tĂ© d ĂȘtre rĂ©versiblement commutable de certaines protĂ©ines fluorescenteshomologues Ă la GFP ouvre un vaste champ d applications possibles: notamment le biostockagede donnĂ©es Ă haute densitĂ© et la microscopie Ă super rĂ©solution. Parmi ces protĂ©ines,on trouve plusieurs variantes de la GFP, notamment la protĂ©ine jaune YFP, et des protĂ©inesfluorescentes issues d'espĂšces marines Anthozoaires, comme Dronpa ou Padron. PlusieursĂ©tudes structurales indiquent que ces protĂ©ines fluorescentes photochromiques commutent parisomĂ©risation et protonation couplĂ©es du chromophore. Cependant, la synchronisation entreces deux Ă©vĂ©nements, le dĂ©tail des mĂ©canismes de photo-commutation, et le rĂŽle de ladynamique conformationelle restent incomplĂštement compris. Par l'utilisation combinĂ©e de lacristallographie cinĂ©tique et de la spectroscopie optique in cristallo Ă basse tempĂ©rature, nousavons comparĂ© le comportement des protĂ©ines YFP, Dronpa et IrisFP, et nous avons Ă©tudiĂ© endĂ©tail le mĂ©canisme photo-physique de commutation chez la protĂ©ine Padron. Contrairement Ă Dronpa et IrisFP, la photo-commutation d YFP est plus efficace Ă basse tempĂ©rature qu Ă tempĂ©rature ambiante. Nos rĂ©sultats suggĂšrent que le mĂ©canisme de commutation d YFPn'implique pas de changement conformationel majeur, mais plutĂŽt une protonation photoinduitedu chromophore ne nĂ©cessitant pas d'isomĂ©risation. Au contraire, les Ă©tudes rĂ©alisĂ©essur la protĂ©ine Padron nous ont permis de montrer que, dans ce cas, l isomĂ©risation duchromophore peut se produire indĂ©pendamment de sa protonation, et, Ă©tonnamment, Ă tempĂ©rature cryogĂ©nique. De plus, deux Ă©tats intermĂ©diaires ont pu ĂȘtre caractĂ©risĂ©s au coursdu processus de photo-commutation. La protĂ©ine Padron a permis de mettre Ă jour le premiermarqueur codable gĂ©nĂ©tiquement qui soit efficacement photo-commutable Ă tempĂ©raturecryogĂ©nique.The property to be reversible switchable of some homologues fluorescents protein ofGFP open a large field for possible applications: such as, high-density data bio-storage andsuper-resolution microscopy. Between these proteins, we find several variants of GFP, such asyellow fluorescent protein, YFP, and fluorescents protein from marine Anthozoary species, asDronpa or Padron. Several structural studies suggest that these fluorescent proteins switch viaisomerization coupled with the protonation of the chromophore. However, thesynchronization between these processes, the detail about the photo-switching mechanism,and the role of conformational dynamics remains unclear. In combination of the kineticcrystallography and the optic spectroscopy in cristallo at low temperature, we have comparedthe YFP behavior, Dronpa and IrisFP, and we have studied in detail the photo-physicmechanism of Padron switching. In contrast to Dronpa and IrisFP, the YFP photoswitching ismore efficient at low temperature than at room temperature. Our results suggest that theYFPswitching is not associated to large structural rearrangements, but mostly a photo-inducedprotonation of the chromophore without isomerization. On the contrary, the studies done withPadron allowed us to show, in this case, the chromophore isomerization can be producedindependently of the protonation, at cryo-temperatures. Moreover, two intermediate stateswere revealed in the photo-pathway. Padron fluorescent protein allows to advance the firstgenetically inserted dye, being photo-switchable at cryogenic temperatureSAVOIE-SCD - Bib.Ă©lectronique (730659901) / SudocGRENOBLE1/INP-Bib.Ă©lectronique (384210012) / SudocGRENOBLE2/3-Bib.Ă©lectronique (384219901) / SudocSudocFranceF
Mécanismes de photo-commutation réversible des protéines fluorescentes
The property to be reversible switchable of some homologues fluorescents protein ofGFP open a large field for possible applications: such as, high-density data bio-storage andsuper-resolution microscopy. Between these proteins, we find several variants of GFP, such asyellow fluorescent protein, YFP, and fluorescents protein from marine Anthozoary species, asDronpa or Padron. Several structural studies suggest that these fluorescent proteins switch viaisomerization coupled with the protonation of the chromophore. However, thesynchronization between these processes, the detail about the photo-switching mechanism,and the role of conformational dynamics remains unclear. In combination of the kineticcrystallography and the optic spectroscopy in cristallo at low temperature, we have comparedthe YFP behavior, Dronpa and IrisFP, and we have studied in detail the photo-physicmechanism of Padron switching. In contrast to Dronpa and IrisFP, the YFP photoswitching ismore efficient at low temperature than at room temperature. Our results suggest that theYFPswitching is not associated to large structural rearrangements, but mostly a photo-inducedprotonation of the chromophore without isomerization. On the contrary, the studies done withPadron allowed us to show, in this case, the chromophore isomerization can be producedindependently of the protonation, at cryo-temperatures. Moreover, two intermediate stateswere revealed in the photo-pathway. Padron fluorescent protein allows to advance the firstgenetically inserted dye, being photo-switchable at cryogenic temperatureLa propriĂ©tĂ© dâĂȘtre rĂ©versiblement commutable de certaines protĂ©ines fluorescenteshomologues Ă la GFP ouvre un vaste champ dâapplications possibles: notamment le biostockagede donnĂ©es Ă haute densitĂ© et la microscopie Ă super rĂ©solution. Parmi ces protĂ©ines,on trouve plusieurs variantes de la GFP, notamment la protĂ©ine jaune YFP, et des protĂ©inesfluorescentes issues d'espĂšces marines Anthozoaires, comme Dronpa ou Padron. PlusieursĂ©tudes structurales indiquent que ces protĂ©ines fluorescentes photochromiques commutent parisomĂ©risation et protonation couplĂ©es du chromophore. Cependant, la synchronisation entreces deux Ă©vĂ©nements, le dĂ©tail des mĂ©canismes de photo-commutation, et le rĂŽle de ladynamique conformationelle restent incomplĂštement compris. Par l'utilisation combinĂ©e de lacristallographie cinĂ©tique et de la spectroscopie optique in cristallo Ă basse tempĂ©rature, nousavons comparĂ© le comportement des protĂ©ines YFP, Dronpa et IrisFP, et nous avons Ă©tudiĂ© endĂ©tail le mĂ©canisme photo-physique de commutation chez la protĂ©ine Padron. Contrairement Ă Dronpa et IrisFP, la photo-commutation dâYFP est plus efficace Ă basse tempĂ©rature quâĂ tempĂ©rature ambiante. Nos rĂ©sultats suggĂšrent que le mĂ©canisme de commutation dâYFPn'implique pas de changement conformationel majeur, mais plutĂŽt une protonation photoinduitedu chromophore ne nĂ©cessitant pas d'isomĂ©risation. Au contraire, les Ă©tudes rĂ©alisĂ©essur la protĂ©ine Padron nous ont permis de montrer que, dans ce cas, lâisomĂ©risation duchromophore peut se produire indĂ©pendamment de sa protonation, et, Ă©tonnamment, Ă tempĂ©rature cryogĂ©nique. De plus, deux Ă©tats intermĂ©diaires ont pu ĂȘtre caractĂ©risĂ©s au coursdu processus de photo-commutation. La protĂ©ine Padron a permis de mettre Ă jour le premiermarqueur codable gĂ©nĂ©tiquement qui soit efficacement photo-commutable Ă tempĂ©raturecryogĂ©nique
Serial femtosecond crystallography reveals that photoactivation in a fluorescent protein proceeds via the hula twist mechanism
Chromophore cis/trans photoisomerization is a fundamental process in chemistry and in the activation of many photosensitive proteins. A major task is understanding the effect of the protein environment on the efficiency and direction of this reaction compared to what is observed in the gas and solution phases. In this study, we set out to visualize the hula twist (HT) mechanism in a fluorescent protein, which is hypothesized to be the preferred mechanism in a spatially constrained binding pocket. We use a chlorine substituent to break the twofold symmetry of the embedded phenolic group of the chromophore and unambiguously identify the HT primary photoproduct. Through serial femtosecond crystallography, we then track the photoreaction from femtoseconds to the microsecond regime. We observe signals for the photoisomerization of the chromophore as early as 300 fs, obtaining the first experimental structural evidence of the HT mechanism in a protein on its femtosecond-to-picosecond timescale. We are then able to follow how chromophore isomerization and twisting lead to secondary structure rearrangements of the protein ÎČ-barrel across the time window of our measurements
Computational mechanistic photochemistry: The central role of conical intersections
In this thesis, I review my own contributions in the field of computational photochemistry. This manuscript is written as an introduction to this field of research. It is not intended to be a textbook, as more emphasis has been made on illustrations rather than on methodologies and technical guidelines. In this way, I hope that it will be accessible to a large audience, from undergraduate students to more experienced scientists who would be interested in learning about this fascinating and relatively young field of research