scholarly journals Super-resolution Microscopy of Clickable Amino Acids Reveals the Effects of Fluorescent Protein Tagging on Protein Assemblies

ACS Nano ◽  
2015 ◽  
Vol 9 (11) ◽  
pp. 11034-11041 ◽  
Author(s):  
Ingrid C. Vreja ◽  
Ivana Nikić ◽  
Fabian Göttfert ◽  
Mark Bates ◽  
Katharina Kröhnert ◽  
...  
Keyword(s):  
Amino Acids ◽  
Fluorescent Protein ◽  
Super Resolution ◽  
Protein Assemblies ◽  
Super Resolution Microscopy ◽  
Protein Tagging

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>

Nuclear Pore Scaffold Structure Analyzed by Super-Resolution Microscopy and Particle Averaging

Science ◽  
2013 ◽  
Vol 341 (6146) ◽  
pp. 655-658 ◽  
Author(s):  
Anna Szymborska ◽  
Alex de Marco ◽  
Nathalie Daigle ◽  
Volker C. Cordes ◽  
John A. G. Briggs ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Super Resolution ◽  
Molecular Organization ◽  
Nuclear Pore ◽  
Protein Assemblies ◽  
Large Protein ◽  
Whole Cells ◽  
Molecular Machinery ◽  
Scaffold Structure ◽  
Super Resolution Microscopy

Much of life’s essential molecular machinery consists of large protein assemblies that currently pose challenges for structure determination. A prominent example is the nuclear pore complex (NPC), for which the organization of its individual components remains unknown. By combining stochastic super-resolution microscopy, to directly resolve the ringlike structure of the NPC, with single particle averaging, to use information from thousands of pores, we determined the average positions of fluorescent molecular labels in the NPC with a precision well below 1 nanometer. Applying this approach systematically to the largest building block of the NPC, the Nup107-160 subcomplex, we assessed the structure of the NPC scaffold. Thus, light microscopy can be used to study the molecular organization of large protein complexes in situ in whole cells.

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

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 Bioorthogonal labeling of transmembrane proteins with non-canonical amino acids allows access to masked epitopes in live neurons

2021 ◽  
Author(s):  
Diogo Bessa-Neto ◽  
Alexander Kuhlemann ◽  
Gerti Beliu ◽  
Valeria Pecoraro ◽  
Sören Doose ◽  
...  
Keyword(s):  
Amino Acids ◽  
Brain Slices ◽  
Transmembrane Proteins ◽  
Super Resolution ◽  
Biological Imaging ◽  
Bioorthogonal Labeling ◽  
Resolution Imaging ◽  
Genetic Code Expansion ◽  
Super Resolution Microscopy ◽  
Organotypic Brain Slices

ABSTRACTProgress in biological imaging is intrinsically linked to advances in labeling methods. The explosion in the development of high-resolution and super-resolution imaging calls for new approaches to label targets with small probes. These should allow to faithfully report the localization of the target within the imaging resolution – typically nowadays a few nanometers - and allow access to any epitope of the target, in the native cellular and tissue environment. We report here the development of a complete labeling and imaging pipeline using genetic code expansion and non-canonical amino acids in primary neurons that allows to fluorescently label masked epitopes in target transmembrane proteins in live neurons, both in dissociated culture and organotypic brain slices. This allowed us to image the differential localization of two glutamate receptor auxiliary proteins in complex with their partner with a variety of methods including widefield, confocal, and dSTORM super-resolution microscopy.

scholarly journals Bright ligand-activatable fluorescent protein for high-quality multicolor live-cell super-resolution microscopy

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Jiwoong Kwon ◽  
Jong-Seok Park ◽  
Minsu Kang ◽  
Soobin Choi ◽  
Jumi Park ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Super Resolution ◽  
Live Cell ◽  
High Quality ◽  
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>

Coordinate-Targeted and Coordinate-Stochastic Super-Resolution Microscopy with the Reversibly Switchable Fluorescent Protein Dreiklang

ChemPhysChem ◽  
2014 ◽  
Vol 15 (4) ◽  
pp. 756-762 ◽  
Author(s):  
Nickels A. Jensen ◽  
Johann G. Danzl ◽  
Katrin I. Willig ◽  
Flavie Lavoie-Cardinal ◽  
Tanja Brakemann ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Super Resolution ◽  
Super Resolution Microscopy

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.

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