scholarly journals NMR reveals light-induced changes in the dynamics of a photoswitchable fluorescent protein

2019 ◽  
Author(s):  
N. E. Christou ◽  
I. Ayala ◽  
K. Giandoreggio-Barranco ◽  
M. Byrdin ◽  
V. Adam ◽  
...  
Keyword(s):  
Nmr Spectroscopy ◽  
Environmental Conditions ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Conformational Dynamics ◽  
X Ray ◽  
X Ray Crystallography ◽  
Dynamic View ◽  
Fluorescent State ◽  
Induced Changes

AbstractThe 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 non-fluorescent state. Our results add a dynamic view to the static structures obtained by X-ray crystallography. Including NMR into the analytical toolbox used for 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.SignificancePhoto-transformable Fluorescent Proteins (PTFPs) are essential tools for super-resolution (SR) microscopy. In practical applications, however, researchers often encounter problems when using PTFPs in a particular cellular context, because the environmental conditions (pH, temperature, redox potential, oxygen level, viscosity, …) affect their brightness, photostability, phototransformation kinetics, etc. Rational fluorescent protein engineering exploits the mechanistic information available from structural studies, mainly X-ray crystallography, in order to design new PTFP variants with improved properties for particular applications. Here we apply NMR spectroscopy in solution to investigate the light-induced changes in conformational dynamics of rsFolder, a reversibly switchable fluorescent protein. The dynamic view offered by NMR highlights protein regions that comprise potentially interesting mutation points for future mutagenesis campaigns.

scholarly journals Structural analysis of the bright monomeric yellow-green fluorescent protein mNeonGreen obtained by directed evolution

2016 ◽  
Vol 72 (12) ◽  
pp. 1298-1307 ◽  
Author(s):  
Damien Clavel ◽  
Guillaume Gotthard ◽  
David von Stetten ◽  
Daniele De Sanctis ◽  
Hélène Pasquier ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Directed Evolution ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
X Rays ◽  
Visible Absorption ◽  
X Ray ◽  
X Ray Crystallography ◽  
Green Fluorescent

Until recently, genes coding for homologues of the autofluorescent protein GFP had only been identified in marine organisms from the phyla Cnidaria and Arthropoda. New fluorescent-protein genes have now been found in the phylum Chordata, coding for particularly bright oligomeric fluorescent proteins such as the tetrameric yellow fluorescent proteinlanYFP fromBranchiostoma lanceolatum. A successful monomerization attempt led to the development of the bright yellow-green fluorescent protein mNeonGreen. The structures oflanYFP and mNeonGreen have been determined and compared in order to rationalize the directed evolution process leading from a bright, tetrameric to a still bright, monomeric fluorescent protein. An unusual discolouration of crystals of mNeonGreen was observed after X-ray data collection, which was investigated using a combination of X-ray crystallography and UV–visible absorption and Raman spectroscopies, revealing the effects of specific radiation damage in the chromophore cavity. It is shown that X-rays rapidly lead to the protonation of the phenolate O atom of the chromophore and to the loss of its planarity at the methylene bridge.

scholarly journals Efficient switching of mCherry fluorescence using chemical caging

2017 ◽  
Vol 114 (27) ◽  
pp. 7013-7018 ◽  
Author(s):  
Bas M. C. Cloin ◽  
Elke De Zitter ◽  
Desiree Salas ◽  
Vincent Gielen ◽  
Gert E. Folkers ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Functional Characterization ◽  
Fluorescence Emission ◽  
Total Recovery ◽  
X Ray Crystallography ◽  
Chemically Induced ◽  
Wide Range ◽  
Fluorescent State

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.

scholarly journals Aequorea’s secrets revealed: New fluorescent proteins with unique properties for bioimaging and biosensing

PLoS Biology ◽  
2020 ◽  
Vol 18 (11) ◽  
pp. e3000936
Author(s):  
Gerard G. Lambert ◽  
Hadrien Depernet ◽  
Guillaume Gotthard ◽  
Darrin T. Schultz ◽  
Isabelle Navizet ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Polypeptide Chain ◽  
De Novo ◽  
Fluorescent Proteins ◽  
Transcriptome Assembly ◽  
X Ray ◽  
X Ray Crystallography ◽  
Green Fluorescent ◽  
New Generation

Using mRNA sequencing and de novo transcriptome assembly, we identified, cloned, and characterized 9 previously undiscovered fluorescent protein (FP) homologs from Aequorea victoria and a related Aequorea species, with most sequences highly divergent from A. victoria green fluorescent protein (avGFP). Among these FPs are the brightest green fluorescent protein (GFP) homolog yet characterized and a reversibly photochromic FP that responds to UV and blue light. Beyond green emitters, Aequorea species express purple- and blue-pigmented chromoproteins (CPs) with absorbances ranging from green to far-red, including 2 that are photoconvertible. X-ray crystallography revealed that Aequorea CPs contain a chemically novel chromophore with an unexpected crosslink to the main polypeptide chain. Because of the unique attributes of several of these newly discovered FPs, we expect that Aequorea will, once again, give rise to an entirely new generation of useful probes for bioimaging and biosensing.

[Zn(phen)2(CX3COO)]+, X = H orCl; Influence of X on the Coordination Mode of the Carboxylate Group (phen = 1,10-Phenanthroline)

2005 ◽  
Vol 60 (10) ◽  
pp. 1049-1053 ◽  
Author(s):  
Zeanab Talaei ◽  
Ali Morsali ◽  
Ali R. Mahjoub
Keyword(s):  
Nmr Spectroscopy ◽  
Elemental Analysis ◽  
Coordination Mode ◽  
Bidentate Ligand ◽  
Carboxylate Group ◽  
X Ray ◽  
X Ray Crystallography ◽  
H Nmr ◽  
Coordinate Geometry ◽  
C Nmr

Two new ZnII(phen)2 complexes with trichloroacetate and acetate anions, [Zn(phen)2(CCl3COO)- (H2O)](ClO4) and [Zn(phen)2(CH3COO)](ClO4), have been synthesized and characterized by elemental analysis, IR, 1H NMR, 13C NMR spectroscopy. The single crystal X-ray data of these compounds show the Zn atoms to have six-coordinate geometry. From IR spectra and X-ray crystallography it is established that the coordination of the COO− group is different for trichloroacetate and acetate. The former acts as a monodentate whereas the latter acts as a bidentate ligand.

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