Virgile Adam

Structural Characterization of Irisfp, an Optical Highlighter Undergoing Multiple Photo-Induced Transformations.

Photoactivatable fluorescent proteins (FPs) are powerful fluorescent highlighters in live cell imaging and offer perspectives for optical nanoscopy and the development of biophotonic devices. Two types of photoactivation are currently being distinguished, reversible photoswitching between fluorescent and nonfluorescent forms and irreversible photoconversion. Here, we have combined crystallography and (in crystallo) spectroscopy to characterize the Phe-173-Ser mutant of the tetrameric variant of EosFP, named IrisFP, which incorporates both types of phototransformations. In its green fluorescent state, IrisFP displays reversible photoswitching, which involves cis–trans isomerization of the chromophore. Like its parent protein EosFP, IrisFP also photoconverts irreversibly to a red-emitting state under violet light because of an extension of the conjugated π-electron system of the chromophore, accompanied by a cleavage of the polypeptide backbone. The red form of IrisFP exhibits a second reversible photoswitching process, which may also involve cis–trans isomerization of the chromophore. Therefore, IrisFP displays altogether 3 distinct photoactivation processes. The possibility to engineer and precisely control multiple phototransformations in photoactivatable FPs offers exciting perspectives for the extension of the fluorescent protein toolkit.

A microspectrophotometer for UV-visible absorption and fluorescence studies of protein crystals

Absorption microspectrophotometry has been shown to be of considerable help to probe crystalline proteins containing chromophores, metal centres, or coloured substrates/co-factors. Absorption spectra contribute to the proper interpretation of crystallographic structures, especially when transient intermediate states are studied. Here it is shown that fluorescence microspectrophotometry might also be used for such purposes if endogenous fluorophores are present in the macromolecule or when exogenous fluorophores are added and either bind to the protein or reside in the solvent channels. An off-line microspectrophotometer that is able to perform low-temperature absorption and fluorescence spectroscopy on crystals mounted in cryo-loops is described. One-shot steady-state emission spectra of outstanding quality were routinely collected from several samples. In some cases, crystals with optical densities that are too low or too high for absorption studies can still be tackled with fluorescence microspectrophotometry. The technique may be used for simple controls such as checking the presence, absence or redox state of a fluorescent substrate/co-factor. Potential applications in the field of kinetic crystallography are numerous. In addition, the possibility to probe key physico-chemical parameters of the crystal, such as temperature, pH or solvent viscosity, could trigger new studies in protein dynamics.

Rational Design of Photoconvertible and Biphotochromic Fluorescent Proteins for Advanced Microscopy Applications

Advanced fluorescence imaging, including subdiffraction microscopy, relies on fluorophores with controllable emission properties. Chief among these fluorophores are the photoactivatable fluorescent proteins capable of reversible on/off photoswitching or irreversible green-to-red photoconversion. IrisFP was recently reported as the first fluorescent protein combining these two types of phototransformations. The introduction of this protein resulted in new applications such as super-resolution pulse-chase imaging. However, the spectroscopic properties of IrisFP are far from being optimal and its tetrameric organization complicates its use as a fusion tag. Here, we demonstrate how four-state optical highlighting can be rationally introduced into photoconvertible fluorescent proteins and develop and characterize a new set of such enhanced optical highlighters derived from mEosFP and Dendra2. We present in particular NijiFP, a promising new fluorescent protein with photoconvertible and biphotochromic properties that make it ideal for advanced fluorescence-based imaging applications.

Structure of superoxide reductase bound to ferrocyanide and active site expansion upon X-ray-induced photo-reduction.

Some sulfate-reducing and microaerophilic bacteria rely on the enzyme superoxide reductase (SOR) to eliminate the toxic superoxide anion radical (O2*-). SOR catalyses the one-electron reduction of O2*- to hydrogen peroxide at a nonheme ferrous iron center. The structures of Desulfoarculus baarsii SOR (mutant E47A) alone and in complex with ferrocyanide were solved to 1.15 and 1.7 A resolution, respectively. The latter structure, the first ever reported of a complex between ferrocyanide and a protein, reveals that this organo-metallic compound entirely plugs the SOR active site, coordinating the active iron through a bent cyano bridge. The subtle structural differences between the mixed-valence and the fully reduced SOR-ferrocyanide adducts were investigated by taking advantage of the photoelectrons induced by X-rays. The results reveal that photo-reduction from Fe(III) to Fe(II) of the iron center, a very rapid process under a powerful synchrotron beam, induces an expansion of the SOR active site.

Structural Basis of Enhanced Photoconversion Yield in Green Fluorescent Protein-Like Protein Dendra2.

Dendra2 is an engineered, monomeric GFP-like protein that belongs to a subclass of fluorescent proteins undergoing irreversible photoconversion from a green- to a red-emitting state upon exposure to purple-blue light. This photoinduced process occurs only in the neutral state of the chromophore and is known to result from backbone cleavage accompanied by an extension of the delocalized pi-electron system. We have measured the X-ray structure of the green species of Dendra2 and performed a comprehensive characterization of the optical absorption and fluorescence properties of the protein in both its green and red forms. The structure, which is very similar to those reported for the closely related proteins EosFP and Kaede, revealed a local structural change involving mainly Arg66 and a water molecule W4, which are part of a charged and hydrogen-bonded cluster of amino acids and water molecules next to the chromophore. Unlike in EosFP and Kaede, Arg66 of Dendra2 does not contribute to negative charge stabilization on the imidazolinone ring by hydrogen bonding to the imidazolinone carbonyl. This structural change may explain the blue shift of the absorption and emission bands, as well as the markedly higher pKs of the hydroxyphenyl moiety of the chromophore, which were determined as 7.1 and 7.5 for the green and red species, respectively. The action spectrum of photoconversion coincides with the absorption band of the neutral species. Consequently, its 20-fold enhancement in Dendra2 at physiological pH accounts for the higher photoconversion yield of this protein as compared to EosFP.

Reversible Photoswitching in Fluorescent Proteins: A Mechanistic View

Phototransformable fluorescent proteins (FPs) have received considerable attention in recent years, because they enable many new exciting modalities in fluorescence microscopy and biotechnology. On illumination with proper actinic light, phototransformable FPs are amenable to long‐lived transitions between various fluorescent or nonfluorescent states, resulting in processes known as photoactivation, photoconversion, or photoswitching. Here, we review the subclass of photoswitchable FPs with a mechanistic perspective. These proteins offer the widest range of practical applications, including reversible high‐density data bio‐storage, photochromic FRET, and super‐resolution microscopy by either point‐scanning, structured illumination, or single molecule‐based wide‐field approaches. Photoswitching can be engineered to occur with high contrast in both Hydrozoan and Anthozoan FPs and typically results from a combination of chromophore cis‐trans isomerization and protonation change. However, other switching schemes based on, for example, chromophore hydration/dehydration have been discovered, and it seems clear that ever more performant variants will be developed in the future.

Advances in spectroscopic methods for biological crystals. 1. Fluorescence lifetime measurements

Synchrotrons are now producing thousands of macromolecular structures each year. The need for complementary techniques available on site has progressively emerged, either to assess the relevance of the structure of a protein or to monitor changes that may occur during X-ray diffraction data collection. Microspectrophotometers in the UV-visible absorbance or fluorescence mode have evolved over the past few decades to become the instruments of choice to perform such tests. Described here are recent improvements to the microspectrophotometer of the so-called Cryobench laboratory located at the European Synchrotron Radiation Facility, Grenoble, France. Optical and mechanical properties have been enhanced so as to record better spectra on smaller samples. A device has been implemented to measure the signal decay of fluorescent samples, either in the crystalline or in the solution state. Recording of the fluorescence lifetime in addition to the steady-state fluorescence emission spectrum allows precise monitoring of the fluorescent sample under study. The device consists of an adaptation of a commercially available time-correlated single-photon-counting (TCSPC) system. A method to record and analyze series of TCSPC histograms, e.g. collected as a function of temperature, is described. To validate the instruments, fluorescence lifetimes of fluorescent small molecules or proteins in the crystalline or solution state, at room and cryo temperatures, have been measured. Lifetimes of a number of fluorescent proteins of the GFP family were generally found to be shorter in crystals than in solution, and slightly longer at cryo temperatures than at ambient temperature. The possibility of performing fluorescence lifetime measurements on crystals at synchrotron facilities widens the variety of spectroscopic techniques complementing X-ray diffraction on macromolecular crystallography beamlines.

Data storage based on photochromic and photoconvertible fluorescent proteins

The recent discovery of photoconvertible and photoswitchable fluorescent proteins (PCFPs and RSFPs, respectively) that can undergo photoinduced changes of their absorption/emission spectra opened new research possibilities in subdiffraction microscopy and optical data storage. Here we demonstrate the proof-of-principle for read only and rewritable data storage both in 2D and 3D, using PCFPs and RSFPs. The irreversible burning of information was achieved by photoconverting from green to red defined areas in a layer of the PCFP Kaede. Data were also written and erased several times in layers of the photochromic fluorescent protein Dronpa. Using IrisFP, which combines the properties of PCFPs and RSFPs, we performed the first encoding of data in four colours using only one type of fluorescent protein. Finally, three-dimensional optical data storage was demonstrated using three mutants of EosFP (d1EosFP, mEosFP and IrisFP) in their crystalline form. Two-photon excitation allowed the precise addressing of regions of interest (ROIs) within the three-dimensional crystalline matrix without excitation of out-of-focus optical planes. Hence, this contribution highlights several data storage schemes based on the remarkable properties of PCFPs/RSFPs.

From EosFP to mIrisFP: structure-based development of advanced photoactivatable marker proteins of the GFP-family

Fluorescent proteins from the GFP family have become indispensable imaging tools in life sciences research. In recent years, a wide variety of these proteins were discovered in non-bioluminescent anthozoa. Some of them feature exciting new properties, including the possibility to change their fluorescence quantum yield and/or color by irradiating with light of specific wavelengths. These photoactivatable fluorescent proteins enable many interesting applications including pulse-chase experiments and super-resolution imaging. In this review, we discuss the development of advanced variants, using a structure-function based, molecular biophysics approach, of the photoactivatable fluorescent protein EosFP, which can be photoconverted from green to red fluorescence by ~400 nm light. A variety of applications are presented that demonstrate the versatility of these marker proteins in live-cell imaging.

Phototransformable fluorescent proteins: Future challenges

In fluorescence microscopy, the photophysical properties of the fluorescent markers play a fundamental role. The beauty of phototransformable fluorescent proteins (PTFPs) is that some of these properties can be precisely controlled by light. A wide range of PTFPs have been developed in recent years, including photoactivatable, photoconvertible and photoswitchable fluorescent proteins. These smart labels triggered a plethora of advanced fluorescence methods to scrutinize biological cells or organisms dynamically, quantitatively and with unprecedented resolution. Despite continuous improvements, PTFPs still suffer from limitations, and mechanistic questions remain as to how these proteins precisely work.