Development of a green reversibly photoswitchable variant of Eos fluorescent protein with fixation resistance

2021 ◽  
pp. mbc.E21-01-0044
Author(s):  
Mitsuo Osuga ◽  
Tamako Nishimura ◽  
Shiro Suetsugu
Keyword(s):  
Single Molecule ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Excitation Light ◽  
Antibody Staining ◽  
Aldehyde Fixation ◽  
Green Fluorescent ◽  
Super Resolution Microscopy

Super-resolution microscopy determines the localization of fluorescent proteins with high precision, beyond the diffraction limit of light. Super-resolution microscopic techniques include photoactivated localization microscopy (PALM), which can localize a single protein by the stochastic activation of its fluorescence. In the determination of single-molecule localization by PALM, the number of molecules that can be analyzed per image is limited. Thus, many images are required to reconstruct the localization of numerous molecules in the cell. However, most fluorescent proteins lose their fluorescence upon fixation. Here, we combined the amino acid substitutions of two Eos protein derivatives, Skylan-S and mEos4b, which are a green reversibly photoswitchable fluorescent protein (RSFP) and a fixation-resistant green-to-red photo-convertible fluorescent protein, respectively, resulting in the fixation-resistant Skylan-S (frSkylan-S), a green RSFP. The frSkylan-S protein is inactivated by excitation light and re-activated by irradiation with violet light, and retained more fluorescence after aldehyde fixation than Skylan-S. The qualities of the frSkylan-S fusion proteins were sufficiently high in PALM observations, as examined using α-tubulin and clathrin light chain. Furthermore, frSkylan-S can be combined with antibody staining for multicolor imaging. Therefore, frSkylan-S is a green fluorescent protein suitable for PALM imaging under aldehyde-fixation conditions.

Structural Evidence of Photoisomerization Pathways in Fluorescent Proteins

2019 ◽  
Author(s):  
Jeffrey Chang ◽  
Matthew Romei ◽  
Steven Boxer
Keyword(s):  
Rational Design ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Unit Cell Size ◽  
Chlorine Substituent ◽  
Gfp Chromophore ◽  
The One ◽  
Green Fluorescent ◽  
Super Resolution Microscopy

<p>Double-bond photoisomerization in molecules such as the green fluorescent protein (GFP) chromophore can occur either via a volume-demanding one-bond-flip pathway or via a volume-conserving hula-twist pathway. Understanding the factors that determine the pathway of photoisomerization would inform the rational design of photoswitchable GFPs as improved tools for super-resolution microscopy. In this communication, we reveal the photoisomerization pathway of a photoswitchable GFP, rsEGFP2, by solving crystal structures of <i>cis</i> and <i>trans</i> rsEGFP2 containing a monochlorinated chromophore. The position of the chlorine substituent in the <i>trans</i> state breaks the symmetry of the phenolate ring of the chromophore and allows us to distinguish the two pathways. Surprisingly, we find that the pathway depends on the arrangement of protein monomers within the crystal lattice: in a looser packing, the one-bond-flip occurs, whereas in a tighter packing (7% smaller unit cell size), the hula-twist occurs.</p><p> </p><p> </p><p> </p><p> </p><p> </p><p> </p> <p> </p>

Expression-Enhanced Fluorescent Proteins Based on Enhanced Green Fluorescent Protein for Super-resolution Microscopy

ACS Nano ◽  
2015 ◽  
Vol 9 (10) ◽  
pp. 9528-9541 ◽  
Author(s):  
Sam Duwé ◽  
Elke De Zitter ◽  
Vincent Gielen ◽  
Benjamien Moeyaert ◽  
Wim Vandenberg ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Green Fluorescent ◽  
Super Resolution Microscopy

Structural Evidence of Photoisomerization Pathways in Fluorescent Proteins

2019 ◽  
Author(s):  
Jeffrey Chang ◽  
Matthew Romei ◽  
Steven Boxer
Keyword(s):  
Rational Design ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Unit Cell Size ◽  
Chlorine Substituent ◽  
Gfp Chromophore ◽  
The One ◽  
Green Fluorescent ◽  
Super Resolution Microscopy

<p>Double-bond photoisomerization in molecules such as the green fluorescent protein (GFP) chromophore can occur either via a volume-demanding one-bond-flip pathway or via a volume-conserving hula-twist pathway. Understanding the factors that determine the pathway of photoisomerization would inform the rational design of photoswitchable GFPs as improved tools for super-resolution microscopy. In this communication, we reveal the photoisomerization pathway of a photoswitchable GFP, rsEGFP2, by solving crystal structures of <i>cis</i> and <i>trans</i> rsEGFP2 containing a monochlorinated chromophore. The position of the chlorine substituent in the <i>trans</i> state breaks the symmetry of the phenolate ring of the chromophore and allows us to distinguish the two pathways. Surprisingly, we find that the pathway depends on the arrangement of protein monomers within the crystal lattice: in a looser packing, the one-bond-flip occurs, whereas in a tighter packing (7% smaller unit cell size), the hula-twist occurs.</p><p> </p><p> </p><p> </p><p> </p><p> </p><p> </p> <p> </p>

scholarly journals Application of SNAP-Tag in Expansion Super-Resolution Microscopy Using DNA Oligostrands

2021 ◽  
Vol 9 ◽  
Author(s):  
Longfang Yao ◽  
Li Zhang ◽  
Yiyan Fei ◽  
Liwen Chen ◽  
Lan Mi ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
New Technology ◽  
Super Resolution ◽  
Target Protein ◽  
Substrate Molecule ◽  
Label System ◽  
Actual Target ◽  
Super Resolution Microscopy

Expansion super-resolution technology is a new technology developed in recent years. It anchors the dye on the hydrogel and the dye expands with the expansion of the hydrogel so that a super-resolution map can be obtained under an ordinary microscope. However, by labeling the target protein with a first antibody and secondary antibody, the distance between the fluorescent group and the actual target protein is greatly increased. Although fluorescent proteins can also be used for expansion super-resolution to reduce this effect, the fluorescent protein is often destroyed during sample preparation. To solve this problem, we developed a novel label system for expansion microscopy, based on a DNA oligostrand linked with a fluorescent dye, acrylamide group (linker), and benzoylguanine (BG, a small substrate molecule for SNAP-tag). This protocol greatly reduced the error between the position of fluorescent group and the actual target protein, and also reduced loss of the fluorescent group during sample preparation.

scholarly journals Electrostatic control of photoisomerization pathways in proteins

Science ◽  
2020 ◽  
Vol 367 (6473) ◽  
pp. 76-79 ◽  
Author(s):  
Matthew G. Romei ◽  
Chi-Yun Lin ◽  
Irimpan I. Mathews ◽  
Steven G. Boxer
Keyword(s):  
Protein Design ◽  
Fluorescence Quantum Yield ◽  
Fluorescent Protein ◽  
Super Resolution ◽  
Molecular Devices ◽  
Electrostatic Effects ◽  
Generalized Framework ◽  
Electrostatic Control ◽  
Green Fluorescent ◽  
Super Resolution Microscopy

Rotation around a specific bond after photoexcitation is central to vision and emerging opportunities in optogenetics, super-resolution microscopy, and photoactive molecular devices. Competing roles for steric and electrostatic effects that govern bond-specific photoisomerization have been widely discussed, the latter originating from chromophore charge transfer upon excitation. We systematically altered the electrostatic properties of the green fluorescent protein chromophore in a photoswitchable variant, Dronpa2, using amber suppression to introduce electron-donating and electron-withdrawing groups to the phenolate ring. Through analysis of the absorption (color), fluorescence quantum yield, and energy barriers to ground- and excited-state isomerization, we evaluate the contributions of sterics and electrostatics quantitatively and demonstrate how electrostatic effects bias the pathway of chromophore photoisomerization, leading to a generalized framework to guide protein design.

scholarly journals Fluorophore-labelled RNA aptamers to common protein tags as super-resolution imaging reagents.

2020 ◽  
Author(s):  
Juan Wang ◽  
Avtar Singh ◽  
Abdullah Ozer ◽  
Warren R Zipfel
Keyword(s):  
Fluorescent Protein ◽  
Super Resolution ◽  
Rna Aptamers ◽  
Cellular Structures ◽  
Intracellular Proteins ◽  
Resolution Imaging ◽  
Green Fluorescent ◽  
Protein Tags ◽  
Super Resolution Microscopy ◽  
Conventional Antibody

Developing labelling methods that densely and specifically label targeted cellular structures is critically important for centroid localization-based super-resolution microscopy. Being easy and inexpensive to produce in the laboratory and of relatively small size, RNA aptamers have potential as a substitute for conventional antibody labelling. By using aptamers selected against common protein tags - GFP (green fluorescent protein) in this case - we demonstrate labelling methods using dSTORM-compatible fluorophores for STORM and hybridizable imager strands for DNA-PAINT super-resolution optical imaging of any cellular proteins fused to the aptamer binding target. We show that we can label both extracellular and intracellular proteins for super-resolution imaging, and that the method in particular, offers some interesting advantages for live cell super-resolution imaging of plasma membrane proteins.

Photoactivated structural dynamics of fluorescent proteins

2012 ◽  
Vol 40 (3) ◽  
pp. 531-538 ◽  
Author(s):  
Dominique Bourgeois ◽  
Aline Regis-Faro ◽  
Virgile Adam
Keyword(s):  
Protein Dynamics ◽  
Structural Dynamics ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Simulation Tools ◽  
X Ray Crystallography ◽  
Hybrid Approaches ◽  
Actinic Light ◽  
Green Fluorescent

Proteins of the GFP (green fluorescent protein) family have revolutionized life sciences because they allow the tagging of biological samples in a non-invasive genetically encoded way. ‘Phototransformable’ fluorescent proteins, in particular, have recently attracted widespread interest, as their fluorescence state can be finely tuned by actinic light, a property central to the development of super-resolution microscopy. Beyond microscopy applications, phototransformable fluorescent proteins are also exquisite tools to investigate fundamental protein dynamics. Using light to trigger processes such as photoactivation, photoconversion, photoswitching, blinking and photobleaching allows the exploration of the conformational landscape in multiple directions. In the present paper, we review how structural dynamics of phototransformable fluorescent proteins can be monitored by combining X-ray crystallography, in crystallo optical spectroscopy and simulation tools such as quantum chemistry/molecular mechanics hybrid approaches. Besides their usefulness to rationally engineer better performing fluorescent proteins for nanoscopy and other biotechnological applications, these investigations provide fundamental insights into protein dynamics.

scholarly journals Saturated excitation of fluorescent proteins for subdiffraction-limited imaging of living cells in three dimensions

2013 ◽  
Vol 3 (5) ◽  
pp. 20130007 ◽  
Author(s):  
Masahito Yamanaka ◽  
Kenta Saito ◽  
Nicholas I. Smith ◽  
Satoshi Kawata ◽  
Takeharu Nagai ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Living Cells ◽  
Three Dimensions ◽  
Fluorescence Signal ◽  
Optical Transfer Function ◽  
Excitation Light ◽  
Optical Transfer ◽  
Green Fluorescent ◽  
Nonlinear Components

We report, for the first time, the saturated excitation (SAX) of fluorescent proteins for subdiffraction-limited imaging of living cells in three-dimensions. To achieve saturation, a bright yellow and green fluorescent protein (Venus and EGFP) that exhibits a strong nonlinear fluorescence response to the high excitation intensity at the laser focus is used. Harmonic demodulation of the fluorescence signal produced by a modulated excitation light extracts the nonlinear fluorescence signals. After constructing the image from the nonlinear components, we obtain fluorescence images of living cells with spatial resolution beyond the diffraction limit. We also applied linear deconvolution to SAX microscopy and found it effective in further enhancing the contrast of small intracellular structures in the SAX image, confirming the expansion of the optical transfer function in SAX microscopy.

scholarly journals Internal Conversion of the Anionic GFP Chromophore: In and Out of the I-twisted S1/S0 Conical Intersection Seam

2021 ◽  
Author(s):  
Nanna Holmgaard List ◽  
Chey Marcel Jones ◽  
Todd J. Martínez
Keyword(s):  
Internal Conversion ◽  
Fluorescent Protein ◽  
Dynamical Behavior ◽  
Super Resolution ◽  
Ultrafast Relaxation ◽  
Gfp Chromophore ◽  
Adiabatic Dynamics ◽  
Dynamics Simulations ◽  
Green Fluorescent ◽  
Super Resolution Microscopy

<p>The functional diversity of the green fluorescent protein (GFP) family is intimately connected to the interplay between competing photo-induced transformations of the chromophore motif, anionic <i>p</i>-hydroxybenzylidene-2,3-dimethylimidazolinone (HBDI<sup>–</sup>). Its propensity to undergo <i>Z/E</i> photoisomerization following excitation to the S<sub>1</sub>(pp<sup>*</sup>) state is of particular importance for super-resolution microscopy and emerging opportunities in optogenetics. However, key dynamical aspects of this process and its range of tunability still remain elusive. Here, we investigate the internal conversion behavior intrinsic to HBDI<sup>–</sup> with focus on competing deactivation pathways, rate and yield of photoisomerization. Based on non-adiabatic dynamics simulations, we confirm that non-selective progress along the two bridge-torsional (i.e., phenolate, P, or imidazolinone, I) pathways can account for the three decay constants reported experimentally, leading to competing ultrafast relaxation along the I-twisted pathway and S<sub>1 </sub>trapping along the P-torsion. The majority of the population (~70%) is transferred to S<sub>0</sub> in the vicinity of two near-symmetry-related minima on the I-twisted intersection seam (MECI-Is). Despite their reactant-biased topographies, our account of inertial effects suggests that isomerization not only occurs as a thermal process on the vibrationally hot ground state but also as a direct photoreaction with a total quantum yield of ~40%.</p><p>By comparing the non-adiabatic dynamics to a photoisomerization committor analysis, we provide a detailed mapping of the intrinsic photoreactivity and dynamical behavior of the two MECI-Is. Our work offers new insight into the internal conversion process of HBDI<sup>–</sup> that enlightens principles for the design of chromophore derivatives and protein variants with improved photoswitching properties.</p>

scholarly journals Choosing the right label for single-molecule tracking in live bacteria: Side-by-side comparison of photoactivatable fluorescent protein and Halo tag dyes

2018 ◽  
Author(s):  
Nehir Banaz ◽  
Jarno Mäkelä ◽  
Stephan Uphoff
Keyword(s):  
Single Molecule ◽  
High Speed ◽  
Cell Function ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Super Resolution ◽  
Single Molecule Tracking ◽  
Advantages And Disadvantages ◽  
Fluorescent Ligands ◽  
The Right

AbstractVisualizing and quantifying molecular motion and interactions inside living cells provides crucial insight into the mechanisms underlying cell function. This has been achieved by super-resolution localization microscopy and single-molecule tracking in conjunction with photoactivatable fluorescent proteins. An alternative labelling approach relies on genetically-encoded protein tags with cell-permeable fluorescent ligands which are brighter and less prone to photobleaching than fluorescent proteins but require a laborious labelling process. Either labelling method is associated with significant advantages and disadvantages that should be taken into consideration depending on the microscopy experiment planned. Here, we describe an optimised procedure for labelling Halo-tagged proteins in live Escherichia coli cells. We provide a side-by-side comparison of Halo tag with different fluorescent ligands against the popular photoactivatable fluorescent protein PAmCherry. Using test proteins with different intracellular dynamics, we evaluated fluorescence intensity, background, photostability, and single-molecule localization and tracking results. Capitalising on the brightness and extended spectral range of fluorescent Halo ligands, we also demonstrate high-speed and dual-colour single-molecule tracking.

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