Elke De Zitter

Green-to-red photoconvertible Dronpa mutant for multimodal super-resolution fluorescence microscopy

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.

Structural Characterization of the Complex between Hen Egg-White Lysozyme and Zr(IV) -Substituted Keggin Polyoxometalate as Artificial Protease.

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.

Protein‐Assisted Formation and Stabilization of Catalytically Active Polyoxometalate Species

The effect of the protein environment on the formation and stabilization of an elusive catalytically active polyoxometalate (POM) species, K6 [Hf(α2 -P2 W17 O61 )] (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.

Efficient switching of mCherry fluorescence using chemical caging

Significance In contrast to diffraction-limited microscopy, superresolution microscopy highly depends on the used fluorescent label. However, introducing a new label with suitable dynamics is not always straightforward. Here we describe how mCherry, a frequently used fluorescent protein in conventional microscopy, can be used for superresolution microscopy via a new caging mechanism involving the addition of β-mercaptoethanol. Moreover, we investigate the structural mechanism behind this chemical caging, using X-ray crystallography, NMR spectroscopy, and ab initio quantum mechanical calculations. These show that the mechanism is twofold: β-mercaptoethanol adds covalently to the protein’s chromophore, whereas it also acts as a reducing agent for the chromophore. Fluorophores with dynamic or controllable fluorescence emission have become essential tools for advanced imaging, such as superresolution imaging. These applications have driven the continuing development of photoactivatable or photoconvertible labels, including genetically encoded fluorescent proteins. These new probes work well but require the introduction of new labels that may interfere with the proper functioning of existing constructs and therefore require extensive functional characterization. In this work we show that the widely used red fluorescent protein mCherry can be brought to a purely chemically induced blue-fluorescent state by incubation with β-mercaptoethanol (βME). The molecules can be recovered to the red fluorescent state by washing out the βME or through irradiation with violet light, with up to 80% total recovery. We show that this can be used to perform single-molecule localization microscopy (SMLM) on cells expressing mCherry, which renders this approach applicable to a very wide range of existing constructs. We performed a detailed investigation of the mechanism underlying these dynamics, using X-ray crystallography, NMR spectroscopy, and ab initio quantum-mechanical calculations. We find that the βME-induced fluorescence quenching of mCherry occurs both via the direct addition of βME to the chromophore and through βME-mediated reduction of the chromophore. These results not only offer a strategy to expand SMLM imaging to a broad range of available biological models, but also present unique insights into the chemistry and functioning of a highly important class of fluorophores.

Trapping a long-lived dark state in photoconvertible fluorescent protein mEos4b

In PhotoActivation Localization Microscopy (PALM), the most popular type of fluorescent proteins labels are green-to-red photoconvertible fluorescent proteins (PCFPs). As photoconversion is irreversible, these markers are in principle especially suited for “quantitative” PALM (qPALM) where molecular copy numbers and stoichiometry at the level of individual protein complexes and structures are investigated (1). In practice however, qPALM suffers from fluorescent protein blinking, a process in which the FP tends to repeatedly enter a reversible dark state. This blinking causes photoconverted (red) molecules to be potentially counted more than once, and may also compete with photoconversion of green molecules. Blinking is thus highly detrimental for accurate quantitative studies. In order to design a PCFP with reduced or even suppressed blinking, the associated mechanisms must first be understood (2,3).