Structural Heterogeneity in a Phototransformable Fluorescent Protein Impacts its Photochemical Properties.

Photoconvertible fluorescent proteins (PCFP) are important cellular markers in advanced imaging modalities such as photoactivatable localization microscopy (PALM). However, their complex photophysical and photochemical behavior hampers applications such as quantitative and single-particle-tracking PALM. This work employs multidimensional NMR combined with ensemble fluorescence measurements to show that the popular mEos4b in its Green state populates two conformations (A and B), differing in side-chain protonation of the conserved residues E212 and H62,  altering the hydrogen-bond network in the chromophore pocket. The interconversion (protonation/deprotonation) between these two states, which occurs on the minutes time scale in the dark, becomes strongly accelerated in the presence of UV light, leading to a population shift. This work shows that the reversible photoswitching and Green-to-Red photoconversion properties differ between the A and B states. The chromophore in the A-state photoswitches more efficiently and is proposed to be more prone to photoconversion, while the B-state shows a higher level of photobleaching. Altogether, this data highlights the central role of conformational heterogeneity in fluorescent protein photochemistry.

Photophysical Studies at Cryogenic Temperature Reveal a Novel Photoswitching Mechanism of rsEGFP2.

Single-molecule localization microscopy (SMLM) at cryogenic temperature opens new avenues to investigate intact biological samples at the nanoscale and perform cryo-correlative studies. Genetically encoded fluorescent proteins (FPs) are markers of choice for cryo-SMLM, but their reduced conformational flexibility below the glass-transition temperature hampers efficient cryo-photoswitching. We investigated cryo-switching of rsEGFP2, one of the most efficient reversibly switchable fluorescent proteins at ambient temperature due to facile cis-trans isomerization of the chromophore. UV-visible microspectrophotometry and X-ray crystallography revealed a completely different switching mechanism at ∼110 K. At this cryogenic temperature, on-off photoswitching involves the formation of two off-states in cis conformation with blue-shifted absorption relative to that of the trans protonated chromophore populated at ambient temperature. Only one of these off-states can be switched back to the fluorescent on-state by 405 nm light, while both of them are sensitive to UV light at 355 nm. Superior recovery to the fluorescent on-state by 355 nm light was confirmed at the single-molecule level. This suggests, as also shown by simulations, that employing 355 nm light in cryo-SMLM experiments using rsEGFP2 and possibly other FPs could improve the effective labeling efficiency achievable with this technique. The rsEGFP2 photoswitching mechanism discovered in this work adds to the panoply of known switching mechanisms in fluorescent proteins.

Rational Control of Off-State Heterogeneity in a Photoswitchable Fluorescent Protein Provides Switching Contrast Enhancement.

Reversibly photoswitchable fluorescent proteins are essential markers for advanced biological imaging, and optimization of their photophysical properties underlies improved performance and novel applications. Here we establish a link between photoswitching contrast, one of the key parameters that dictate the achievable resolution in nanoscopy applications, and chromophore conformation in the non-fluorescent state of rsEGFP2, a widely employed label in REversible Saturable OpticaL Fluorescence Transitions (RESOLFT) microscopy. Upon illumination, the cis chromophore of rsEGFP2 isomerizes to two distinct off-state conformations, trans1 and trans2, located on either side of the V151 side chain. Reducing or enlarging the side chain at this position (V151A and V151L variants) leads to single off-state conformations that exhibit higher and lower switching contrast, respectively, compared to the rsEGFP2 parent. The combination of structural information obtained by serial femtosecond crystallography with high-level quantum chemical calculations and with spectroscopic and photophysical data determined in vitro suggests that the changes in switching contrast arise from blue- and red-shifts of the absorption bands associated to trans1 and trans2, respectively. Thus, due to elimination of trans2, the V151A variants of rsEGFP2 and its superfolding variant rsFolder2 display a more than two-fold higher switching contrast than their respective parent proteins, both in vitro and in E. coli cells. The application of the rsFolder2-V151A variant is demonstrated in RESOLFT nanoscopy. Our study rationalizes the connection between structural and photophysical chromophore properties and suggests a means to rationally improve fluorescent proteins for nanoscopy applications.

Disentangling Chromophore States in a Reversibly Switchable Green Fluorescent Protein: Mechanistic Insights from NMR Spectroscopy.

The photophysical properties of fluorescent proteins, including phototransformable variants used in advanced microscopy applications, are influenced by the environmental conditions in which they are expressed and used. Rational design of improved fluorescent protein markers requires a better understanding of these environmental effects. We demonstrate here that solution NMR spectroscopy can detect subtle changes in the chemical structure, conformation, and dynamics of the photoactive chromophore moiety with atomic resolution, providing such mechanistic information. Studying rsFolder, a reversibly switchable green fluorescent protein, we have identified four distinct configurations of its p-HBI chromophore, corresponding to the cis and trans isomers, with each one either protonated (neutral) or deprotonated (anionic) at the benzylidene ring. The relative populations and interconversion kinetics of these chromophore species depend on sample pH and buffer composition that alter in a complex way the strength of H-bonds that contribute in stabilizing the chromophore within the protein scaffold. We show in particular the important role of histidine-149 in stabilizing the neutral trans chromophore at intermediate pH values, leading to ground-state cis-trans isomerization with a peculiar pH dependence. We discuss the potential implications of our findings on the pH dependence of the photoswitching contrast, a critical parameter in nanoscopy applications.

Supramolecular assembly of the Escherichia coli LdcI upon acid stress.

Pathogenic and commensal bacteria often have to resist the harsh acidity of the host stomach. The inducible lysine decarboxylase LdcI buffers the cytosol and the local extracellular environment to ensure enterobacterial survival at low pH. Here, we investigate the acid stress-response regulation of Escherichia coli LdcI by combining biochemical and biophysical characterization with negative stain and cryoelectron microscopy (cryo-EM) and wide-field and superresolution fluorescence imaging. Due to deleterious effects of fluorescent protein fusions on native LdcI decamers, we opt for three-dimensional localization of nanobody-labeled endogenous wild-type LdcI in acid-stressed E. coli cells and show that it organizes into distinct patches at the cell periphery. Consistent with recent hypotheses that in vivo clustering of metabolic enzymes often reflects their polymerization as a means of stimulus-induced regulation, we show that LdcI assembles into filaments in vitro at physiologically relevant low pH. We solve the structures of these filaments and of the LdcI decamer formed at neutral pH by cryo-EM and reveal the molecular determinants of LdcI polymerization, confirmed by mutational analysis. Finally, we propose a model for LdcI function inside the enterobacterial cell, providing a structural and mechanistic basis for further investigation of the role of its supramolecular organization in the acid stress response.

Mechanistic Investigations of Green mEos4b Reveal a Dynamic Long-Lived Dark State.

Green-to-red photoconvertible fluorescent proteins (PCFPs) are key players in advanced microscopy schemes such as photoactivated localization microscopy (PALM). Whereas photoconversion and red-state blinking in PCFPs have been studied intensively, their green-state photophysical behavior has received less attention. Yet dark states in green PCFPs can become strongly populated in PALM schemes and exert an indirect but considerable influence on the quality of data recorded in the red channel. Furthermore, green-state photoswitching in PCFPs can be used directly for PALM and has been engineered to design highly efficient reversibly switchable fluorescent proteins (RSFPs) amenable to various nanoscopy schemes. Here, we demonstrate that green mEos4b efficiently switches to a long-lived dark state through cis-trans isomerization of its chromophore, as do most RSFPs. However, by combining kinetic crystallography, molecular dynamics simulations, and Raman spectroscopy, we find that the dark state in green mEos4b is much more dynamic than that seen in switched-off green IrisFP, a biphotochromic PCFP engineered from the common EosFP parent. Our data suggest that H-bonding patterns maintained by the chromophore in green PCFPs and RSFPs in both their on- and off-states collectively control photoswitching quantum yields. The reduced number of H-bonds maintained by the dynamic dark chromophore in green mEos4b thus largely accounts for the observed lower switching contrast as compared to that of IrisFP. We also compare the long-lived dark states reached from green and red mEos4b, on the basis of their X-ray structures and Raman signatures. Altogether, these data provide a unifying picture of the complex photophysics of PCFPs and RSFPs.

Photoswitching mechanism of a fluorescent protein revealed by time-resolved crystallography and transient absorption spectroscopy.

Reversibly switchable fluorescent proteins (RSFPs) serve as markers in advanced fluorescence imaging. Photoswitching from a non-fluorescent off-state to a fluorescent on-state involves trans-to-cis chromophore isomerization and proton transfer. Whereas excited-state events on the ps timescale have been structurally characterized, conformational changes on slower timescales remain elusive. Here we describe the off-to-on photoswitching mechanism in the RSFP rsEGFP2 by using a combination of time-resolved serial crystallography at an X-ray free-electron laser and ns-resolved pump-probe UV-visible spectroscopy. Ten ns after photoexcitation, the crystal structure features a chromophore that isomerized from trans to cis but the surrounding pocket features conformational differences compared to the final on-state. Spectroscopy identifies the chromophore in this ground-state photo-intermediate as being protonated. Deprotonation then occurs on the µs timescale and correlates with a conformational change of the conserved neighbouring histidine. Together with a previous excited-state study, our data allow establishing a detailed mechanism of off-to-on photoswitching in rsEGFP2.

NMR Reveals Light-Induced Changes in the Dynamics of a Photoswitchable Fluorescent Protein.

The availability of fluorescent proteins with distinct phototransformation properties is crucial for a wide range of applications in advanced fluorescence microscopy and biotechnology. To rationally design new variants optimized for specific applications, a detailed understanding of the mechanistic features underlying phototransformation is essential. At present, little is known about the conformational dynamics of fluorescent proteins at physiological temperature and how these dynamics contribute to the observed phototransformation properties. Here, we apply high-resolution NMR spectroscopy in solution combined with in situ sample illumination at different wavelengths to investigate the conformational dynamics of rsFolder, a GFP-derived protein that can be reversibly switched between a green fluorescent state and a nonfluorescent state. Our results add a dynamic view to the static structures obtained by x-ray crystallography. Including a custom-tailored NMR toolbox in fluorescent protein research provides new opportunities for investigating the effect of mutations or changes in the environmental conditions on the conformational dynamics of phototransformable fluorescent proteins and their correlation with the observed photochemical and photophysical properties.

Mechanistic investigation of mEos4b reveals a strategy to reduce track interruptions in sptPALM.

Green-to-red photoconvertible fluorescent proteins repeatedly enter dark states, causing interrupted tracks in single-particle-tracking localization microscopy (sptPALM). We identified a long-lived dark state in photoconverted mEos4b that results from isomerization of the chromophore and efficiently absorbs cyan light. Addition of weak 488-nm light swiftly reverts this dark state to the fluorescent state. This strategy largely eliminates slow blinking and enables the recording of longer tracks in sptPALM with minimum effort.

Chromophore twisting in the excited state of a photoswitchable fluorescent protein captured by time-resolved serial femtosecond crystallography.

Chromophores absorb light in photosensitive proteins and thereby initiate fundamental biological processes such as photosynthesis, vision and biofluorescence. An important goal in their understanding is the provision of detailed structural descriptions of the ultrafast photochemical events that they undergo, in particular of the excited states that connect chemistry to biological function. Here we report on the structures of two excited states in the reversibly photoswitchable fluorescent protein rsEGFP2. We populated the states through femtosecond illumination of rsEGFP2 in its non-fluorescent off state and observed their build-up (within less than one picosecond) and decay (on the several picosecond timescale). Using an X-ray free-electron laser, we performed picosecond time-resolved crystallography and show that the hydroxybenzylidene imidazolinone chromophore in one of the excited states assumes a near-canonical twisted configuration halfway between the trans and cis isomers. This is in line with excited-state quantum mechanics/molecular mechanics and classical molecular dynamics simulations. Our new understanding of the structure around the twisted chromophore enabled the design of a mutant that displays a twofold increase in its off-to-on photoswitching quantum yield.

Photoswitching of Green mEos2 by Intense 561 nm Light Perturbs Efficient Green-to-Red Photoconversion in Localization Microscopy.

Green-to-red photoconvertible fluorescent proteins (PCFPs) such as mEos2 and its derivatives are widely used in PhotoActivated Localization Microscopy (PALM). However, the complex photophysics of these genetically encoded markers complicates the quantitative analysis of PALM data. Here, we show that intense 561 nm light (∼1 kW/cm2) typically used to localize single red molecules considerably affects the green-state photophysics of mEos2 by populating at least two reversible dark states. These dark states retard green-to-red photoconversion through a shelving effect, although one of them is rapidly depopulated by 405 nm light illumination. Multiple mEos2 switching and irreversible photobleaching is thus induced by yellow/green and violet photons before green-to-red photoconversion occurs, contributing to explain the apparent limited signaling efficiency of this PCFP. Our data reveals that the photophysics of PCFPs of anthozoan origin is substantially more complex than previously thought, and suggests that intense 561 nm laser light should be used with care, notably for quantitative or fast PALM approaches.

Serial Femtosecond Crystallography and Ultrafast Absorption Spectroscopy of the Photoswitchable Fluorescent Protein IrisFP.

Reversibly photoswitchable fluorescent proteins find growing applications in cell biology, yet mechanistic details, in particular on the ultrafast photochemical time scale, remain unknown. We employed time-resolved pump-probe absorption spectroscopy on the reversibly photoswitchable fluorescent protein IrisFP in solution to study photoswitching from the nonfluorescent (off) to the fluorescent (on) state. Evidence is provided for the existence of several intermediate states on the pico- and microsecond time scales that are attributed to chromophore isomerization and proton transfer, respectively. Kinetic modeling favors a sequential mechanism with the existence of two excited state intermediates with lifetimes of 2 and 15 ps, the second of which controls the photoswitching quantum yield. In order to support that IrisFP is suited for time-resolved experiments aiming at a structural characterization of these ps intermediates, we used serial femtosecond crystallography at an X-ray free electron laser and solved the structure of IrisFP in its on state. Sample consumption was minimized by embedding crystals in mineral grease, in which they remain photoswitchable. Our spectroscopic and structural results pave the way for time-resolved serial femtosecond crystallography aiming at characterizing the structure of ultrafast intermediates in reversibly photoswitchable fluorescent proteins.

Rational design of ultrastable and reversibly photoswitchable fluorescent proteins for super-resolution imaging of the bacterial periplasm.

Phototransformable fluorescent proteins are central to several nanoscopy approaches. As yet however, there is no available variant allowing super-resolution imaging in cell compartments that maintain oxidative conditions. Here, we report the rational design of two reversibly switchable fluorescent proteins able to fold and photoswitch in the bacterial periplasm, rsFolder and rsFolder2. rsFolder was designed by hybridisation of Superfolder-GFP with rsEGFP2, and inherited the fast folding properties of the former together with the rapid switching of the latter, but at the cost of a reduced switching contrast. Structural characterisation of the switching mechanisms of rsFolder and rsEGFP2 revealed different scenarios for chromophore cis-trans isomerisation and allowed designing rsFolder2, a variant of rsFolder that exhibits improved switching contrast and is amenable to RESOLFT nanoscopy. The rsFolders can be efficiently expressed in the E. coli periplasm, opening the door to the nanoscale investigation of proteins localised in hitherto non-observable cellular compartments.

Arginine 66 Controls Dark-State Formation in Green-to-Red Photoconvertible Fluorescent Proteins.

Photoactivated localization microscopy (PALM) is a powerful technique to investigate cellular nanostructures quantitatively and dynamically. However, the use of PALM for molecular counting or single-particle tracking remains limited by the propensity of photoconvertible fluorescent protein markers (PCFPs) to repeatedly enter dark states. By designing the single mutants mEos2-A69T and Dendra2-T69A, we completely swapped the blinking behaviors of mEos2 and Dendra2, two popular PCFPs. We combined X-ray crystallography and single-molecule microscopy to show that blinking in mEos2 and Dendra2 is largely controlled by the orientation of arginine 66, a highly conserved residue in Anthozoan PCFPs. The Arg66 side-chain conformation affects the bleaching and the on-to-off transition quantum yields, as well as the fraction of molecules entering long-lived dark states, resulting in widely different apparent blinking behaviors that largely modulate the efficiency of current blinking correction procedures. The present work provides mechanistic insight into the complex photophysics of Anthozoan PCFPs and will facilitate future engineering of bright and low-blinking variants suitable for PALM.

Remodeling of the Z-Ring Nanostructure during the Streptococcus pneumoniae Cell Cycle Revealed by Photoactivated Localization Microscopy.

UNLABELLED: Ovococci form a morphological group that includes several human pathogens (enterococci and streptococci). Their shape results from two modes of cell wall insertion, one allowing division and one allowing elongation. Both cell wall synthesis modes rely on a single cytoskeletal protein, FtsZ. Despite the central role of FtsZ in ovococci, a detailed view of the in vivo nanostructure of ovococcal Z-rings has been lacking thus far, limiting our understanding of their assembly and architecture. We have developed the use of photoactivated localization microscopy (PALM) in the ovococcus human pathogen Streptococcus pneumoniae by engineering spDendra2, a photoconvertible fluorescent protein optimized for this bacterium. Labeling of endogenously expressed FtsZ with spDendra2 revealed the remodeling of the Z-ring's morphology during the division cycle at the nanoscale level. We show that changes in the ring's axial thickness and in the clustering propensity of FtsZ correlate with the advancement of the cell cycle. In addition, we observe double-ring substructures suggestive of short-lived intermediates that may form upon initiation of septal cell wall synthesis. These data are integrated into a model describing the architecture and the remodeling of the Z-ring during the cell cycle of ovococci. IMPORTANCE: The Gram-positive human pathogen S. pneumoniae is responsible for 1.6 million deaths per year worldwide and is increasingly resistant to various antibiotics. FtsZ is a cytoskeletal protein polymerizing at midcell into a ring-like structure called the Z-ring. FtsZ is a promising new antimicrobial target, as its inhibition leads to cell death. A precise view of the Z-ring architecture in vivo is essential to understand the mode of action of inhibitory drugs (see T. den Blaauwen, J. M. Andreu, and O. Monasterio, Bioorg Chem 55:27-38, 2014, doi:10.1016/j.bioorg.2014.03.007, for a review on FtsZ inhibitors). This is notably true in ovococcoid bacteria like S. pneumoniae, in which FtsZ is the only known cytoskeletal protein. We have used superresolution microscopy to obtain molecular details of the pneumococcus Z-ring that have so far been inaccessible with conventional microscopy. This study provides a nanoscale description of the Z-ring architecture and remodeling during the division of ovococci.

Rational design of enhanced photoresistance in a photoswitchable fluorescent protein.

Fluorescent proteins are particularly susceptible to photobleaching, the permanent loss of fluorescence emission resulting from photodestruction of the chromophore. In the case of Reversibly Switchable Fluorescent Proteins (RSFPs), which can be switched back and forth between a non-fluorescent and a fluorescent state, the achievable number of switching cycles is limited by photobleaching, a process known as photofatigue. Photofatigue has become a crucial limitation in a number of advanced applications based on repeated photoswitching of RSFPs, notably in the field of super-resolution fluorescence microscopy. Here, based on our previous structural investigation of photobleaching mechanisms in IrisFP, an RSFP also capable of green-to-red photoconversion, we present the rational design of a single-mutant IrisFP-M159A that displays considerably enhanced photostability. The results suggest that, under moderate illumination intensities, photobleaching of IrisFP-like Anthozoan fluorescent proteins such as EosFP, Dendra or Dronpa derivatives is mainly driven by an oxygen-dependent mechanism resulting in the irreversible sulfoxidation of methionine 159. The photofatigue decay profiles of IrisFP and its photoresistant mutant IrisFP-M159A were investigated in different experimental conditions, in vitro and in cellulo. Although the performance of the mutant was found to be always superior, the results showed switching behaviors strongly dependent on the nanoenvironment. Thus, in general, assessment of photostability and switching properties of RSFPs should be carried out in real experimental conditions.

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.

In cellulo evaluation of phototransformation quantum yields in fluorescent proteins used as markers for single-molecule localization microscopy.

Single-molecule localization microscopy of biological samples requires a precise knowledge of the employed fluorescent labels. Photoactivation, photoblinking and photobleaching of phototransformable fluorescent proteins influence the data acquisition and data processing strategies to be used in (Fluorescence) Photoactivation Localization Microscopy ((F)-PALM), notably for reliable molecular counting. As these parameters might depend on the local environment, they should be measured in cellulo in biologically relevant experimental conditions. Here, we measured phototransformation quantum yields for Dendra2 fused to actin in fixed mammalian cells in typical (F)-PALM experiments. To this aim, we developed a data processing strategy based on the clustering optimization procedure proposed by Lee et al (PNAS 109, 17436-17441, 2012). Using simulations, we estimated the range of experimental parameters (molecular density, molecular orientation, background level, laser power, frametime) adequate for an accurate determination of the phototransformation yields. Under illumination at 561 nm in PBS buffer at pH 7.4, the photobleaching yield of Dendra2 fused to actin was measured to be (2.5 ± 0.4) × 10(-5), whereas the blinking-off yield and thermally-activated blinking-on rate were measured to be (2.3 ± 0.2) × 10(-5) and 11.7 ± 0.5 s-1, respectively. These phototransformation yields differed from those measured in poly-vinyl alcohol (PVA) and were strongly affected by addition of the antifading agent 1,4-diazabicyclo[2.2.2]octane (DABCO). In the presence of DABCO, the photobleaching yield was reduced 2-fold, the blinking-off yield was decreased more than 3-fold, and the blinking-on rate was increased 2-fold. Therefore, DABCO largely improved Dendra2 photostability in fixed mammalian cells. These findings are consistent with redox-based bleaching and blinking mechanisms under (F)-PALM experimental conditions. Finally, the green-to-red photoconversion quantum yield of Dendra2 was estimated to be (1.4 ± 0.6) × 10(-5) in cellulo under 405 nm illumination.

Structural basis of photoswitching in fluorescent proteins.

Fluorescent proteins have revolutionized life sciences because they allow noninvasive and highly specific labeling of biological samples. The subset of "phototransformable" fluorescent proteins recently attracted a widespread interest, as their fluorescence state can be modified upon excitation at defined wavelengths. The fluorescence emission of Reversibly Switchable Fluorescent Proteins (RSFPs), in particular, can be repeatedly switched on and off. RSFPs enable many new exciting modalities in fluorescence microscopy and biotechnology, including protein tracking, photochromic Förster Resonance Energy Transfer, super-resolution microscopy, optogenetics, and ultra-high-density optical data storage. Photoswitching in RSFPs typically results from chromophore cis-trans isomerization accompanied by a protonation change, but other switching schemes based on, e.g., chromophore hydration/dehydration have also been discovered. In this chapter, we review the main structural features at the basis of photoswitching in RSFPs.

Phototransformable fluorescent proteins: which one for which application?

In these last two decades , fluorescent proteins (FPs) have become highly valued imaging tools for cell biology, owing to their compatibility with living samples, their low levels of invasiveness and the possibility to specifically fuse them to a variety of proteins of interest. Remarkably, the recent development of phototransformable fluorescent proteins (PTFPs) has made it possible to conceive optical imaging experiments that were unimaginable only a few years ago. For example, it is nowadays possible to monitor intra- or intercellular trafficking, to optically individualize single cells in tissues or to observe single molecules in live cells. The tagging specificity brought by these genetically encoded highlighters leads to constant progress in the engineering of increasingly powerful, versatile and non-cytotoxic FPs. This review is focused on the recent developments of PTFPs and highlights their contribution to studies within cells, tissues and even living organisms. The aspects of single-molecule localization microscopy, intracellular tracking of photoactivated molecules, applications of PTFPs in biotechnology/optobiology and complementarities between PTFPs and other microscopy techniques are particularly discussed.