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.

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 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.

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 rsEGFP2 enables fast RESOLFT nanoscopy of living cells

eLife ◽  
2012 ◽  
Vol 1 ◽  
Author(s):  
Tim Grotjohann ◽  
Ilaria Testa ◽  
Matthias Reuss ◽  
Tanja Brakemann ◽  
Christian Eggeling ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Super Resolution ◽  
Living Cells ◽  
Low Light ◽  
Light Levels ◽  
Sequential Sample ◽  
Green Fluorescent ◽  
Super Resolution Microscopy

The super-resolution microscopy called RESOLFT relying on fluorophore switching between longlived states, stands out by its coordinate-targeted sequential sample interrogation using low light levels. While RESOLFT has been shown to discern nanostructures in living cells, the reversibly photoswitchable green fluorescent protein (rsEGFP) employed in these experiments was switched rather slowly and recording lasted tens of minutes. We now report on the generation of rsEGFP2 providing faster switching and the use of this protein to demonstrate 25–250 times faster recordings.

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>

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.

Dramatic reduction in fluorescence quantum yield in mutants of Green Fluorescent Protein due to fast internal conversion

1998 ◽  
Vol 237 (1-2) ◽  
pp. 183-193 ◽  
Author(s):  
Andreas D Kummer ◽  
Christian Kompa ◽  
Harald Lossau ◽  
Florian Pöllinger-Dammer ◽  
Maria E Michel-Beyerle ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Quantum Yield ◽  
Fluorescence Quantum Yield ◽  
Internal Conversion ◽  
Fluorescent Protein ◽  
Dramatic Reduction ◽  
Green Fluorescent

scholarly journals Computational prediction of the tolerance to amino-acid deletion in green-fluorescent protein

2016 ◽  
Author(s):  
Eleisha L. Jackson ◽  
Stephanie J. Spielman ◽  
Claus O. Wilke
Keyword(s):  
Green Fluorescent Protein ◽  
Protein Structure ◽  
Amino Acid ◽  
Protein Design ◽  
Fluorescent Protein ◽  
Computational Prediction ◽  
Single Amino Acid ◽  
Dimensional Structure ◽  
Amino Acid Deletion ◽  
Green Fluorescent

AbstractProteins evolve through two primary mechanisms: substitution, where mutations alter a protein’s amino-acid sequence, and insertions and deletions (indels), where amino acids are either added to or removed from the sequence. Protein structure has been shown to influence the rate at which substitutions accumulate across sites in proteins, but whether structure similarly constrains the occurrence of indels has not been rigorously studied. Here, we investigate the extent to which structural properties known to covary with protein evolutionary rates might also predict protein tolerance to indels. Specifically, we analyze a publicly available dataset of single–amino-acid deletion mutations in enhanced green fluorescent protein (eGFP) to assess how well the functional effect of deletions can be predicted from protein structure. We find that weighted contact number (WCN), which measures how densely packed a residue is within the protein’s three-dimensional structure, provides the best single predictor for whether eGFP will tolerate a given deletion. We additionally find that using protein design to explicitly model deletions results in improved predictions of functional status when combined with other structural predictors. Our work suggests that structure plays fundamental role in constraining deletions at sites in proteins, and further that similar biophysical constraints influence both substitutions and deletions. This study therefore provides a solid foundation for future work to examine how protein structure influences tolerance of more complex indel events, such as insertions or large deletions.

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