Electron and Hydrogen Atom Transfers in the Hydride Carrier Protein EmoB

In this article, we investigate the mechanism of hydride transfer taking place within the EmoB protein of the <i>Mesorhizobium</i> species. The reaction involves the net transfer of one proton and two electrons from a reduced flavin mononucleotide (FMN) cofactor, which is anchored in the protein scaffold, to a diffusible oxidized FMN cofactor, both being held together by π-stacking interactions. To analyze the formal hydride transfer in terms of more elementary steps, electron transfer (ET), and hydrogen atom transfers (HAT), we employ a combination of classical molecular dynamics simulations and hybrid constrained Density Functional Theory/Molecular Mechanics (cDFT/MM) energy calculations to build the free energy profiles, for the ET before and after HAT occurs between the flavins. The main outcomes of our study are first to highlight the role of the protein in stabilizing the π-stacked FMN dimer and second to reveal the coupling between the ET and HAT. Before HAT has taken place, ET is unfavorable by 8 kcal/mol and become favorable by 8 kcal/mol after HAT. Our simulations show that such a coupling is not present for the analogous process in water (ET is almost athermal). This suggests a functional role for the protein matrix to ensure EmoB a role of hydride carrier in the <i>Mesorhizobium</i> species

Structural Evidence for a Two-Regime Photobleaching Mechanism in a Reversibly Switchable Fluorescent Protein

Photobleaching, the irreversible photodestruction of a chromophore, severely limits the use of fluorescent proteins (FPs) in optical microscopy. Yet, the mechanisms that govern photobleaching remain poorly understood. In Reversibly Switchable Fluorescent Proteins (RSFPs), a class of FPs that can be repeatedly photoswitched between nonfluorescent and fluorescent states, photobleaching limits the achievable number of switching cycles, a process known as photofatigue. We investigated the photofatigue mechanisms in the protein IrisFP using combined X-ray crystallography, optical <i>in crystallo</i> spectroscopy, mass spectrometry and modeling approaches. At laser-light intensities typical of conventional wide-field fluorescence microscopy, an oxygen-dependent photobleaching pathway was evidenced. Structural modifications induced by singlet-oxygen production within the chromophore pocket revealed the oxidation of two sulfur-containing residues, Met159 and Cys171, locking the chromophore in a nonfluorescent protonated state. At laser-light intensities typical of localization-based nanoscopy (>0.1 kW/cm²), a completely different, oxygen-independent photobleaching pathway was found to take place. The conserved Glu212 underwent decarboxylation concomitantly with an extensive rearrangement of the H-bond network around the chromophore, and an sp²-to-sp³ hybridization change of the carbon atom bridging the chromophore cyclic moieties was observed. This two-regime photobleaching mechanism is likely to be a common feature in RSFPs from Anthozoan species, which typically share high structural and sequence identity with IrisFP. In addition, our results suggest that, when such FPs are used, the illumination conditions employed in localization-based super-resolution microscopy might generate less cytotoxicity than those of standard wide-field microscopy at constant absorbed light-dose. Finally, our data will facilitate the rational design of FPs displaying enhanced photoresistance

Structural Evidence for a Two-Regime Photobleaching Mechanism in a Reversibly Switchable Fluorescent Protein

Photobleaching, the irreversible photodestruction of a chromophore, severely limits the use of fluorescent proteins (FPs) in optical microscopy. Yet, the mechanisms that govern photobleaching remain poorly understood. In Reversibly Switchable Fluorescent Proteins (RSFPs), a class of FPs that can be repeatedly photoswitched between nonfluorescent and fluorescent states, photobleaching limits the achievable number of switching cycles, a process known as photofatigue. We investigated the photofatigue mechanisms in the protein IrisFP using combined X-ray crystallography, optical <i>in crystallo</i> spectroscopy, mass spectrometry and modeling approaches. At laser-light intensities typical of conventional wide-field fluorescence microscopy, an oxygen-dependent photobleaching pathway was evidenced. Structural modifications induced by singlet-oxygen production within the chromophore pocket revealed the oxidation of two sulfur-containing residues, Met159 and Cys171, locking the chromophore in a nonfluorescent protonated state. At laser-light intensities typical of localization-based nanoscopy (>0.1 kW/cm²), a completely different, oxygen-independent photobleaching pathway was found to take place. The conserved Glu212 underwent decarboxylation concomitantly with an extensive rearrangement of the H-bond network around the chromophore, and an sp²-to-sp³ hybridization change of the carbon atom bridging the chromophore cyclic moieties was observed. This two-regime photobleaching mechanism is likely to be a common feature in RSFPs from Anthozoan species, which typically share high structural and sequence identity with IrisFP. In addition, our results suggest that, when such FPs are used, the illumination conditions employed in localization-based super-resolution microscopy might generate less cytotoxicity than those of standard wide-field microscopy at constant absorbed light-dose. Finally, our data will facilitate the rational design of FPs displaying enhanced photoresistance

The Single T65S Mutation Generates Brighter Cyan Fluorescent Proteins with Increased Photostability and pH Insensitivity

<div><p>Cyan fluorescent proteins (CFP) derived from <em>Aequorea victoria</em> GFP, carrying a tryptophan-based chromophore, are widely used as FRET donors in live cell fluorescence imaging experiments. Recently, several CFP variants with near-ultimate photophysical performances were obtained through a mix of site-directed and large scale random mutagenesis. To understand the structural bases of these improvements, we have studied more specifically the consequences of the single-site T65S mutation. We find that all CFP variants carrying the T65S mutation not only display an increased fluorescence quantum yield and a simpler fluorescence emission decay, but also show an improved pH stability and strongly reduced reversible photoswitching reactions. Most prominently, the Cerulean-T65S variant reaches performances nearly equivalent to those of mTurquoise, with QY  = 0.84, an almost pure single exponential fluorescence decay and an outstanding stability in the acid pH range (pK_1/2 = 3.6). From the detailed examination of crystallographic structures of different CFPs and GFPs, we conclude that these improvements stem from a shift in the thermodynamic balance between two well defined configurations of the residue 65 hydroxyl. These two configurations differ in their relative stabilization of a rigid chromophore, as well as in relaying the effects of Glu222 protonation at acid pHs. Our results suggest a simple method to greatly improve numerous FRET reporters used in cell imaging, and bring novel insights into the general structure-photophysics relationships of fluorescent proteins.</p> </div

pH dependence of the average fluorescence lifetime of purified CFP variants.

<p>Average lifetimes were determined at 20°C from integration of the corresponding lifetime distributions. Solid lines are for eye guidance only.</p

Alternate configurations of threonine 65 in the crystallographic structures of ECFP.

<p>Amino-acids and water molecules interacting with the residue 65 hydroxyl are shown, according to the structures of Lelimousin et al (2WSN, solid, grey) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049149#pone.0049149-Lelimousin1" target="_blank">[40]</a> and Bae et al (1OXD, transparent, red) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049149#pone.0049149-Bae1" target="_blank">[39]</a>. Protein structures were aligned along their main chain backbones, water oxygens are shown as spheres and major H-bonds as dashed lines.</p

Reversible photobleaching of purified CFP variants.

<p>(A) Reversible bleaching kinetics performed on agarose beads labeled with purified CFPs. After prior equilibration in the dark, sudden and constant illumination at 0.2 W/cm2 was applied for less than 15 sec, while camera images were taken every 200 ms. Illumination was then stopped, and, after a minimum of 3 min in the dark, a short series of fluorescence images was collected to check for reversibility. Continuous lines are best fits to the model F^norm =  y₀+y₁t+y₂ exp(−t/τ_Rev) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049149#pone.0049149.s014" target="_blank">Text S1</a>). (B) Amplitudes of the reversible photobleaching responses of the CFP variants.</p

Synchrotron radiation circular dichroism on purified CFP variants at neutral and acid pHs.

<p>Comparison of the SRCD spectra of ECFP at pH 7.4 (dashed line) and different CFP variants at pH 2.5 (solid lines). The SRCD spectra were recorded at 25°C.</p

pH dependence of the spectral properties of purified ECFP.

<p>(<b>A</b>) Absorption spectra normalized to unit maximum absorbance, and (<b>B</b>) emission spectra normalized to unit surface. The spectra corresponding to 50% fluorescence intensity loss (pH = pK_1/2) is represented by a continuous red line. The absorption spectrum represented by a dashed gray line corresponds to the first acid pH at which a typical denaturated spectrum is observed.</p

Spectral properties of purified CFP variants at neutral pH.

<p>Absorption (dashed lines) and emission (solid lines) spectra were normalized to maximum of the chromophore band. Emission spectra were recorded with excitation at 420 nm.</p