Advanced imaging techniques crucially depend on the labels used. In this work, we present the structure-guided design of a fluorescent protein that displays both reversibly photochromic and green-to-red photoconversion behavior. We first designed ffDronpa, a mutant of the photochromic fluorescent protein Dronpa that matures up to three times faster while retaining its interesting photochromic features. Using a combined evolutionary and structure-driven rational design strategy, we developed a green-to-red photoconvertible ffDronpa mutant, called pcDronpa, and explored different optimization strategies that resulted in its improved version, pcDronpa2. This fluorescent probe combines a high brightness with low photobleaching and photoblinking. We herein show that, despite its tetrameric nature, pcDronpa2 allows for multimodal subdiffraction imaging by sequentially imaging a given sample using both super-resolution fluctuation imaging and localization microscopy.
The effect of the protein environment on the formation and stabilization of an elusive catalytically active polyoxometalate (POM) species, K [Hf(α -P W O )] (1), is reported. In the co-crystal of hen egg-white lysozyme (HEWL) with 1, the catalytically active monomeric species is observed, originating from the dimeric 1:2 POM form, while it is intrinsically unstable under physiological pH conditions. The protein-assisted dissociation of the dimeric POM was rationalized by means of DFT calculations. The dissociation process is unfavorable in bulk water, but becomes favorable in the protein-POM complex due to the low dielectric response at the protein surface. The crystal structure shows that the monomeric form is stabilized by electrostatic and water-mediated hydrogen bonding interactions with the protein. It interacts at three distinct sites, close to the aspartate-containing hydrolysis sites, demonstrating high selectivity towards peptide bonds containing this residue.
Smart fluorophores", such as reversibly switchable fluorescent proteins (RSFPs), are crucial for advanced fluorescence imaging. However, only a limited number of such labels is available and many display reduced biological performance compared to more classical variants.We present the development of robustly photoswitchable variants of EGFP, named rsGreens, that display up to 30-fold higher fluorescence in E. coli colonies grown at 37°C and more than 4-fold higher fluorescence when expressed in HEK293T cells compared to their ancestor protein rsEGFP. This enhancement is not due to an intrinsic increase in the fluorescence brightness of the probes, but rather due to enhanced expression levels that allow many more probe molecules to be functional at any given time. We developed rsGreens displaying a range of photoswitching kinetics and show how these can be used for multi-modal diffraction-unlimited fluorescence imaging such as pcSOFI and RESOLFT, achieving a spatial resolution of ~70 nm. By determining the first ever crystal structures of a negative reversibly switchable FP derived from Aequorea victoria in both the "on"-and "off"-conformation we were able to confirm the presence of a cis-trans isomerization and provide further insights into the mechanisms underlying the photochromism. Our work demonstrates that genetically encoded "smart fluorophores" can be readily optimized for biological performance, and provides a practical strategy for developing maturation-and stability-enhanced photochromic fluorescent proteins.KEYWORDS: fluorescent proteins, reversible photoswitching, super-resolution fluorescence microscopy, SOFI, RESOLFT, crystal structure determination, rsEGFP, superfolder Fluorescent proteins (FPs) enable the minimally-invasive labeling of intracellular structures in live systems. 1 The discovery and development of "smart photoactive FPs", 2,3 with features such as irreversible photoactivation and photoconversion, or reversible photoswitching, allowed the development of diffraction-unlimited imaging techniques such as (f)PALM 4,5 ((fluorescence) photoactivated localization microscopy), RESOLFT 6 (reversible saturable optical fluorescence transitions) and (pc)SOFI 7,8 ((photochromic) stochastic optical fluctuation imaging). These techniques strongly rely on the performance of the fluorophores and considerable efforts have therefore been dedicated to create optimized "smart labels". 9 This is exemplified by the continuous optimization and diversification of the EosFP family, 10-15 or the development of Dronpa 16 mutants with different or added photophysical properties. [17][18][19][20][21][22] Probes that combine multiple "smart" behaviors have also been engineered. [23][24][25] On the whole, however, the general acceptance of the FP-based "smart labels" has not quite risen up to the high expectations set by the many applications they enable. In some cases this is due to concerns surrounding the biological compatibility of the labels, meaning that the label may interfere with the functioning of the syst...
Successful co-crystallization of a noncovalent complex between hen egg-white lysozyme (HEWL) and the monomeric Zr(IV) -substituted Keggin polyoxometalate (POM) (Zr1 K1), (Et2 NH2)3 [Zr(PW11 O39)] (1), has been achieved, and its single-crystal X-ray structure has been determined. The dimeric Zr(IV) -substituted Keggin-type polyoxometalate (Zr1 K2), (Et2 NH2)10 [Zr(PW11 O39 )2] (2), has been previously shown to exhibit remarkable selectivity towards HEWL hydrolysis. The reported X-ray structure shows that the hydrolytically active Zr(IV) -substituted Keggin POM exists as a monomeric species. Prior to hydrolysis, this monomer interacts with HEWL in the vicinity of the previously identified cleavage sites found at Trp28-Val29 and Asn44-Arg45, through water-mediated H-bonding and electrostatic interactions. Three binding sites are observed at the interface of the negatively charged Keggin POM and the positively charged regions of HEWL at: 1) Gly16, Tyr20, and Arg21; 2) Asn44, Arg45, and Asn46; and 3) Arg128.
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 significantly longer tracks in sptPALM with minimum effort. Main textFluorescent proteins (FPs) and in particular green-to-red photoconvertible fluorescent proteins (PCFPs) have become indispensable tools for advanced imaging such as singlemolecule localization microscopy (SMLM) or single-particle tracking photoactivated localization microscopy (sptPALM). Both techniques are however limited by blinking, a process in which the fluorophores stochastically enter reversible dark states. PCFPs display blinking on multiple timescales, arising from different underlying photochemical processes. 1 Fluorescence intermittencies shorter than the typical exposure times used in these imaging methodologies (~tens of milliseconds), such as caused by intersystem crossing to the triplet state, reduce the apparent brightness of the label. Intermittencies longer than the exposure time, in contrast, can cause severe complications such as multiple counting of target molecules in quantitative SMLM and interruptions of single-molecule tracks in sptPALM. 2 However, the mechanistic origin of long-lived dark states in PCFPs has remained unclear and there is presently no strategy to eliminate these. We set out to investigate the nature of long-lived fluorescence intermittencies in photoconverted (red) mEos4b, 3 one of the latest probes in a series of highly popular greento-red PCFPs. Individual molecules of mEos4b were immobilized in a polyacrylamide (PAA) matrix, converted to the red emissive state using 405-nm illumination, and the fluorescence emission visualized in time using a sensitive widefield microscope. The single-molecule fluorescence traces (Fig. 1A) displayed reversible and long-lived intermittencies, which we identified as the blinking giving rise to interpretation difficulties in SMLM and sptPALM. Histograms of the intermittency duration (Supplementary Fig. 1) revealed the presence of at least two dark states, as previously reported in mEos2 or Dendra2 4,5 (Supplementary Note 1). While the shorter-lived dark state was insensitive to the intensity of the employed 561-nm illumination (Supplementary Figure 2), the rate at which the longer-lived dark state returned to the emissive state increased with the illumination intensity, reaching a saturation regime at ~0.8 s -1 above a power density of ~ 1.5 kW/cm² (Fig. 1B), indicating a sensitivity to light.
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