IrisFP: the first biphotochromic fluorescent protein
We developed a new protein, called IrisFP, which will help scientists to monitor the spatio-temporal dynamics of proteins using super-resolution optical microscopy. The results, raise exciting prospects for nanoscopy and biophotonics.
Nanoscopy is an emerging field in microscopy that allows samples to be imaged at spatial resolutions on the order of a few tens of nanometers, considerably higher than that possible by traditional optical microscopy.
One set of nanoscopic techniques makes use of fluorescent proteins that are derived from the natural protein GFP and have fluorescent properties which can be altered in a controlled manner.
Many structural biology groups are trying to improve these techniques by developing a new generation of fluorescent proteins. Some of these proteins have the property of being "photocommutable": they can be switched on or off at will. Others are capable of photoconversion : their colour can be altered by exciting them with laser light.
This PNAS study reports the development of IrisFP, which combines both properties. Using ESRF X-rays, we determined the protein’s atomic structure and characterized each of its colour-states. IrisFP is a highly versatile tool which promises to considerably advance microscopy techniques. By genetically fusing IrisFP to a protein of interest, scientists will be able to monitor the protein’s movements within the cell at unprecedented spatial and temporal resolution.
Besides microscopy, the development of new fluorescent probes raises exciting prospects for nanotechnology. Potential future applications include the development of high-density mass storage media that exploit changes in the colour of crystals of these proteins, allowing a large amount of information to be stored in a nanometric-sized structure.
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.
How do photoconvertible fluorescent proteins change their colour?
Fluorescent proteins of the GFP family are the object of intense study due to their inherent bioluminescence, and have proved to be excellent markers for cellular imaging. In the last few years, "photoactivatable" fluorescent proteins have been developed whose fluorescence properties change as a function of their illumination conditions.
These proteins are crucial to the new "super-resolution" fluorescence imaging methods that permit images of living cells to be obtained at nanometre resolution. One of the most popular photoactivatable proteins in nanoscopy is EosFP. This protein normally fluoresces in green, but when illuminated with violet light its fluorescence changes to red. This "photoconversion" process involves the rupture of the peptide chain next to the chromophore and formation of an enlarged conjugated system, but its mechanism has proved elusive.
Starting from X-ray crystallographic structures of the green and red forms of EosFP, we have employed QC/MM simulation methods to investigate possible reaction pathways and have been able to propose a mechanism for the photoconversion. Absorption of a violet photon by the protein promotes it to its first singlet excited state.
Approximately once in a thousand times, the singlet undergoes a forbidden transition to a triplet state. Once there, a proton transfer occurs that leads to a cascade of events that results in rupture of the peptide backbone, elongation of the conjugated system and red fluorescence.
This work is an advance in our understanding of the function of photoactivatable fluorescent proteins, and could allow the development of variants with improved photophysical properties.
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.
How do 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 GFPs, 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
Shedding 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 (see above). 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 . JACS (2011) 133:18586-9
How 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
Opening 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).
The 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 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).
XFEL structures of photoswitchable fluorescent proteins
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.
Time-resolved and static crystallographic experiments on reversibly switchable fluorescent proteins IrisFP and rsEGFP2 in their on and off state were solved by SFX.
The high-quality structures show no signs of X-ray radiation damage and were determined from a very small amount of crystalline sample for IrisFP using grease as an injection medium. rsEGFP2 crystals were injected in liquid medium and allowed to trap intermediate states along the photoreaction pathway from the trans to the cis isomerization of the chromophore. For this project, the IBS DYNAMOP group used the XFEL at SACLA in Japan and LCLS in the USA and teamed up with scientists the beamlines, the Max-Planck Institute in Heidelberg, the Universities of Lille and Rennes and the ESRF in Grenoble.
As a complement to SFX, time-resolved absorption spectroscopy was used to identify intermediate-states during photoswitching.
Together, our data lay a solid ground for ultra-fast time-resolved SFX at XFELs of photoswitchable fluorescent proteins that, beyond their fascinating photochemistry, are of major importance for advanced nanoscopy, such as super-resolution microscopy.
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