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structure of IrisFPStructural characterization of IrisFP, an optical highlighter undergoing multiple photo-induced transformations
Scientists from the Institut de Biologie Structurale (IBS, a mixed research unit of the CEA, CNRS and Université Joseph Fourier), the ESRF, the University of Ulm (Germany) and the University of Southampton (United Kingdom) have just developed a new fluorescent protein derived from GFP (green fluorescent protein). The new protein, called Iris-FP, will help scientists to monitor the spatio-temporal dynamics of proteins using super-resolution optical microscopy. The results, recently published online by the journal Proceedings of the National Academy of Sciences raises exciting prospects for nanoscopy and biophotonics. Virgile Adam, Mickaël Lelimousin, Susan Boehme, Guillaume Desfonds, Karin Nienhaus, Martin J. Field, Joerg Wiedenmann, Sean McSweeney, G. Ulrich Nienhaus & Dominique Bourgeois, "Structural characterization of IrisFP, an optical highlighter undergoing multiple photo-induced transformations” PNAS(2008) , 105, 18343-48. |
Eos FP photoconversionA Complex Mechanism Explains How a Fluorescent Protein Changes Colour
Mickael Lelimousin, Virgile Adam, G. Ulrich Nienhaus, Dominique Bourgeois and Martin J. Field. Photoconversion of the Fluorescent Protein EosFP : A Hybrid Potential Simulation Study Reveals Intersystem Crossings. JACS (2009) 131:16814-23. |
IrisFP blinkingHow fluorescent proteins blink
Fluorescent proteins from the GFP family are remarkable markers for cell imaging. Their weak photostability, however, constitutes their principal disadvantage. If one observes under the microscope a single fluorescent molecule (a fluorescent protein, or an organic dye for example), blinking can be immediately noticed : fluorescence is not constant over time, but alternates between bright and dark periods. In the case of GFP’s, the molecular and structural origin of blinking remains mysterious. Excited states reactions can generate a transient loss of fluorescence, such as intersystem crossing to the triplet state, chromophore protonation, or chromophore isomerization. Another possibility consists in photo-induced electron transfer, which results in the production of a radical species that is unstable and nonfluorescent. In this work, we have provided evidence for such a radical species, which was generated by x-rays from the ESRF. By combining crystallography, Raman spectroscopy, and absorption and fluorescence spectroscopy, we could show that the radical state is characterized by a severe distortion of the chromophore, which accounts for the loss of fluorescence. This is the first study showing a fluorescent protein in a transiently off state. This study could allow the development of more photostable variants. The work also highlights the importance of electron transfer reactions in fluorescent proteins. Virgile Adam, Philippe Carpentier, Sebastien Violot, Mickaël Lelimousin, Claudine Darnault, G. Ulrich Nienhaus & Dominique Bourgeois, "Structural Basis of X-ray Induced Photobleaching in a Photoactivatable Green Fluorescent Protein”, J. Am. Chem. Soc., (2009), 131:18063–18065 |
Padron CryoswitchA fluorescent protein for cryo-nanoscopy
Fluorescence super-resolution microscopy (“nanoscopy”) opens up a considerable research field for integrated structural biology. “PALM” nanoscopy relies on the on and off photoswitching of particular fluorescent proteins called “photoactivatable” fluorescent proteins. The photoswitching mechanisms are linked to significant conformational changes of the chromophore and of the protein matrix, which are typically blocked at low temperature. However, the development of a nanoscopy scheme working at cryogenic temperature could open the door to insightful correlative studies with cryo-electronic microscopy. Thus, it is important to develop markers that can photoswitch at low temperature. In this work, we have studied the photoswitching mechanism of the fluorescent protein Padron, by combining X-ray crystallography, spectroscopy, and molecular dynamics simulations. We discovered that Padron is capable of photoswitching at 100 Kelvin via trans-cis isomerization of its chromophore in an essentially rigid protein matrix. Such a large conformational change had never been observed at such a low temperature in a protein. Regis-Faro, Aline ; Carpentier, Philippe ; Jonasson, Gabriella ; Pompidor, Guillaume ; Arcizet, Delphine ; Demachy, Isabelle ; Bourgeois, Dominique. "Low-temperature chromophore isomerization reveals the photoswitching mechanism of the fluorescent protein Padron." Journal of the American Chemical Society (2011) 133:16362-5. |
IrisFP Vis blinkShedding new light on fluorescent proteins’ dark states
All fluorescent markers used in cell imaging “blink”, switching quickly and stochastically between bright (fluorescent) and dark (non-fluorescent) states. In the case of fluorescent proteins, the molecular and structural origin of blinking remains mysterious. By employing a combination of experimental approaches (crystallography/optical spectroscopy), we demonstrated in 2009 that a transiently dark state of the fluorescent protein IrisFP could be induced by X-rays, characterized by a severe distortion of its chromophore (Adam et al, JACS, 2009, 131, 18063). However, in real imaging conditions, the blinking process results from illumination with visible light, not with X-rays. In the present work, simulations based on a hybrid approach combining quantum mechanics and molecular mechanics (QM/MM) suggest that IrisFP can blink in essentially the same way under illumination with visible light or X-rays. The chromophore distortion at the origin of the fluorescence intermittency can be explained by the reversible transfer of a proton from a nearby arginine residue towards the central part (methylene bridge) of the chromophore in a triplet or a radical state. This distortion of the chromophore disrupts transiently its electronic conjugation and hence stops its fluorescence emission. This work is important for the future development of more photostable fluorescent proteins. Arijit Roy, Martin J. Field, Virgile Adam and Dominique Bourgeois. The Nature of Transient Dark States in a Photoactivatable Fluorescent Protein |
IrisFP bleachHow do fluorescent proteins die ?
Fluorescent proteins are widespread markers in cellular imaging, providing a highly flexible toolbox to investigate live cells. Unfortunately, contrary to organic dyes, fluorescent proteins are particularly sensitive to the photobleaching phenomenon, the definitive loss of fluorescence following photo-induced destruction of the chromophore. Photobleaching is particularly problematic in super-resolution microscopy techniques, which are being rapidly developed today, limiting the resolution that can be achieved. By combining kinetic crystallography, optical and Raman spectroscopy, molecular dynamics simulations, mass spectrometry, and super resolution microscopy, we have investigated the photophysical mechanisms leading to photobleaching of the fluorescent protein IrisFP. We have shown that depending on the illumination intensity used for the imaging experiment, two completely different photobleaching mechanisms show up. At low laser intensity, typical of a standard widefield microscopy experiment, an oxygen-dependent mechanism predominates. On the contrary, at high laser intensity, typical of super-resolution microscopy experiments, a redox-dependent mechanism prevails. The first mechanism, which generates reactive oxygen species (ROS) in the cell is thus expected to be more cytotoxic than the second mechanism, which does not generate such species. Thus, this work suggests in a counterintuitive manner that by increasing laser intensity at constant those, less cellular damages would be created. This hypothesis now needs to be experimentally verified. Chenxi Duan, Virgile Adam, Martin Byrdin, Jacqueline Ridard, Sylvie Kieffer-Jacquinot, Cécile Morlot, Delphine Arcizet, Isabelle Demachy & Dominique Bourgeois Structural Evidence for a Two-Regime Photobleaching Mechanism in a Reversibly Switchable Fluorescent Protein J. Am. Chem. Soc. (2013), 135 : 15841−15850 |
arg66The dark side of photoconvertible fluorescent proteins
Photoconvertible fluorescent proteins are markers of choice for PhotoActivated Localization Microscopy (PALM). Notably, these markers allow counting target proteins one by one directly inside cells. Unfortunately, the accuracy of counting is limited by “blinking”, that is, the discontinuous character of light emission by a single fluorescent molecule along time. Indeed, a single molecule that blinks can easily be confounded with an ensemble of distinct molecules that appear successively at the same location. Blinking results from stochastic and reversible transitions between fluorescent and dark states, but the involved mechanisms remain poorly understood. Improving the quantitative analysis of PALM data thus relies on the design of low-blinking variants. By combining X-ray crystallography, optical spectroscopy and PALM microscopy, we discovered that the orientation of a unique, fully conserved, aminoacid located next to the chromophore entirely controls the blinking of photoconvertible fluorescent proteins. The knowledge of the orientation of this aminoacid (arginine 66) is then sufficient to accurately predict blinking properties. This research (that ANR refuses to finance …) brings new knowledge in fundamental photophysics and opens the door to the rational engineering of variants optimized for quantitative PALM. Romain Berardozzi, Virgile Adam, Alexandre Martins & Dominique Bourgeois Arginine 66 Controls Dark-State Formation in Green-to-Red Photoconvertible Fluorescent Proteins Journal of the American Chemical Society 138, 558-565 (2016). |
rsFolderOpening the door to super-resolution microscopy in oxidizing cellular compartments
Various cellular compartments such as the endoplasmic reticulum or the mitochondrial intermembrane space may be considered as "hostile" environments, because particularly oxidizing. This is also the case in the bacterial periplasm, a space of major importance for the understanding of cellular respiration, biofilms formation and antibiotic resistance. When proteins of interest are fused to fluorescent proteins to allow their microscopic observation, the latter, once secreted into the oxidizing environments, are generally unable to fold correctly and thus fluoresce. There is one notable exception, Superfolder GFP, unfortunately unsuitable for super resolution microscopy. In this work, combining biochemistry, crystallography and photophysical studies, we realized the rational engineering of Superfolder-GFP in order to make this marker photoswitchable. To this purpose, we constructed a chimeric protein combining Superfolder-GFP and rsEGFP2, a GFP derivative used for super-resolution in non-oxidizing environments. The result : rsFolder is a new tool is a new tool allowing the observation of oxidizing environments such as the periplasm with resolutions of the order of 70 nm. The development of rsFolder is currently ongoing in order to obtain new generations of even more efficient markers for biologists and to access otherwise unobservable hostile cell territories in super-resolution. El Khatib, M., Martins, A., Bourgeois, D., Colletier, J.-P. & Adam, V. Rational design of ultrastable and reversibly photoswitchable fluorescent proteins for super-resolution imaging of the bacterial periplasm. Scientific Reports 6, 18459 (2016). |
Iris FP SFXSerial femtosecond crystallography of the photoswitchable fluorescent protein IrisFP
Serial femtosecond crystallography (SFX) at an X-ray free electron laser (XFEL) exploits intense X-ray pulses to provide a diffraction pattern before radiation damage destroys the protein crystal. The sample is replenished millions of times and diffraction data collected in a serial way. SFX permits tiny microcrystals to be studied and enables time-resolved studies of proteins in action down to the femtosecond time scale. Serial Femtosecond Crystallography and Ultrafast Absorption Spectroscopy of the Photoswitchable Fluorescent Protein IrisFP. Colletier JP, Sliwa M, Gallat FX, Sugahara M, Guillon V, Schiro G, Coquelle N, Woodhouse J, Roux L, Gotthard G, Royant A, Uriarte LM, Ruckebusch C, Joti Y, Byrdin M, Mizohata E, Nango E, Tanaka T, Tono K, Yabashi M, Adam V, Cammarata M, Schlichting I, Bourgeois D, Weik M (2016) The Journal of Physical Chemistry Letters : 882-887 |
mEos2 Dark states |
rsEGFP2 onswitching by XFEL |
EGFP-Triplet |
mEos4b blinkingA strategy to reduce fluorescence intermittencies in sptPALM |
mEos4b blinking in the green statefluorescent proteins dance in the dark |