scholarly journals A photostable monomeric superfolder GFP

2019 ◽  
Author(s):  
Fernando Valbuena ◽  
Ivy Fizgerald ◽  
Rita L. Strack ◽  
Neal Andruska ◽  
Luke Smith ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Photophysical Properties ◽  
General Purpose ◽  
Adverse Conditions ◽  
Aequorea Victoria ◽  
The Past ◽  
Nonspecific Interactions ◽  
Green Fluorescent ◽  
Partner Proteins ◽  
Protein Tagging

ABSTRACTThe green fluorescent protein GFP from Aequorea victoria has been engineered extensively in the past to generate variants suitable for protein tagging. Early efforts produced the enhanced variant EGFP and its monomeric derivative mEGFP, which have useful photophysical properties, as well as superfolder GFP, which folds efficiently under adverse conditions. We previously generated msGFP, a monomeric superfolder derivative of EGFP. Unfortunately, compared to EGFP, msGFP and other superfolder GFP variants show faster photobleaching. We now describe msGFP2, which retains monomeric superfolder properties while being as photostable as EGFP. msGFP2 contains modified N- and C-terminal peptides that are expected to reduce nonspecific interactions. Compared to EGFP and mEGFP, msGFP2 is less prone to disturbing the functions of certain partner proteins. For general-purpose protein tagging, msGFP2 may be the best available derivative of A. victoria GFP.

A Unified Model for Photophysical and Electro-Optical Properties of Green Fluorescent Proteins

2019 ◽  
Author(s):  
Chi-Yun Lin ◽  
Matthew Romei ◽  
Luke Oltrogge ◽  
Irimpan Mathews ◽  
Steven Boxer
Keyword(s):  
Driving Force ◽  
Fluorescent Protein ◽  
Charge Transfer Band ◽  
Photophysical Properties ◽  
Polymethine Dyes ◽  
Emission Rates ◽  
Unified Framework ◽  
Underlying Factor ◽  
Green Fluorescent ◽  
Protein Environment

Green fluorescent protein (GFPs) have become indispensable imaging and optogenetic tools. Their absorption and emission properties can be optimized for specific applications. Currently, no unified framework exists to comprehensively describe these photophysical properties, namely the absorption maxima, emission maxima, Stokes shifts, vibronic progressions, extinction coefficients, Stark tuning rates, and spontaneous emission rates, especially one that includes the effects of the protein environment. In this work, we study the correlations among these properties from systematically tuned GFP environmental mutants and chromophore variants. Correlation plots reveal monotonic trends, suggesting all these properties are governed by one underlying factor dependent on the chromophore's environment. By treating the anionic GFP chromophore as a mixed-valence compound existing as a superposition of two resonance forms, we argue that this underlying factor is defined as the difference in energy between the two forms, or the driving force, which is tuned by the environment. We then introduce a Marcus-Hush model with the bond length alternation vibrational mode, treating the GFP absorption band as an intervalence charge transfer band. This model explains all the observed strong correlations among photophysical properties; related subtopics are extensively discussed in Supporting Information. Finally, we demonstrate the model's predictive power by utilizing the additivity of the driving force. The model described here elucidates the role of the protein environment in modulating photophysical properties of the chromophore, providing insights and limitations for designing new GFPs with desired phenotypes. We argue this model should also be generally applicable to both biological and non-biological polymethine dyes.<br>

scholarly journals Structural basis for dual excitation and photoisomerization of the Aequorea victoria green fluorescent protein

1997 ◽  
Vol 94 (6) ◽  
pp. 2306-2311 ◽  
Author(s):  
K. Brejc ◽  
T. K. Sixma ◽  
P. A. Kitts ◽  
S. R. Kain ◽  
R. Y. Tsien ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Structural Basis ◽  
Aequorea Victoria ◽  
Green Fluorescent

scholarly journals Genetically Encoded Photosensitizers as Light-Triggered Antimicrobial Agents

2019 ◽  
Vol 20 (18) ◽  
pp. 4608 ◽  
Author(s):  
Fabienne Hilgers ◽  
Nora Lisa Bitzenhofer ◽  
Yannic Ackermann ◽  
Alina Burmeister ◽  
Alexander Grünberger ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Antimicrobial Agents ◽  
Fluorescent Protein ◽  
Photophysical Properties ◽  
Type I ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
Intracellular Expression ◽  
Global Concern ◽  
Green Fluorescent

Diseases caused by multi-drug resistant pathogens have become a global concern. Therefore, new approaches suitable for treating these bacteria are urgently needed. In this study, we analyzed genetically encoded photosensitizers (PS) related to the green fluorescent protein (GFP) or light-oxygen-voltage (LOV) photoreceptors for their exogenous applicability as light-triggered antimicrobial agents. Depending on their specific photophysical properties and photochemistry, these PSs can produce different toxic ROS (reactive oxygen species) such as O2•− and H2O2 via type-I, as well as 1O2 via type-II reaction in response to light. By using cell viability assays and microfluidics, we could demonstrate differences in the intracellular and extracellular phototoxicity of the applied PS. While intracellular expression and exogenous supply of GFP-related PSs resulted in a slow inactivation of E. coli and pathogenic Gram-negative and Gram-positive bacteria, illumination of LOV-based PSs such as the singlet oxygen photosensitizing protein SOPP3 resulted in a fast and homogeneous killing of these microbes. Furthermore, our data indicate that the ROS type and yield as well as the localization of the applied PS protein can strongly influence the antibacterial spectrum and efficacy. These findings open up new opportunities for photodynamic inactivation of pathogenic bacteria.

scholarly journals The First Mutant of the Aequorea victoria Green Fluorescent Protein That Forms a Red Chromophore†

Biochemistry ◽  
2008 ◽  
Vol 47 (16) ◽  
pp. 4666-4673 ◽  
Author(s):  
Alexander S. Mishin ◽  
Fedor V. Subach ◽  
Ilia V. Yampolsky ◽  
William King ◽  
Konstantin A. Lukyanov ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Aequorea Victoria ◽  
Green Fluorescent

scholarly journals Crystal structure of the cyan fluorescent protein Cerulean-S175G

2016 ◽  
Vol 72 (7) ◽  
pp. 516-522 ◽  
Author(s):  
Sang-wook Park ◽  
Sunghyun Kang ◽  
Tae-Sung Yoon
Keyword(s):  
Crystal Structure ◽  
Crystal Structures ◽  
Conformational Changes ◽  
Fluorescent Protein ◽  
Detailed Comparison ◽  
Cyan Fluorescent Protein ◽  
Double Mutation ◽  
Aequorea Victoria ◽  
Green Fluorescent ◽  
Enhanced Cyan Fluorescent Protein

Enhanced cyan fluorescent protein (ECFP) was derived fromAequorea victoriagreen fluorescent protein (avGFP), notably with S65T/Y66W mutations. Its chromophore consists of a tripeptide comprised of Thr65, Trp66 and Gly67 (TWG) residues, while that ofavGFP consists of a Ser65, Tyr66 and Gly67 (SYG) tripeptide. Cerulean and SCFP3A were derived from ECFP-S72A/H148D (a double mutation) with additional Y145A and S175G mutations, respectively, while Cerulean-S175G has both mutations (Y145A and S175G). The crystal structures of these ECFP variants at neutral pH were reported to adopt two distinct major conformations calledECFPandCerulean. In this study, Cerulean-S175G was revealed to adopt only theCeruleanconformation, while Cerulean has been reported to adopt both theECFPand theCeruleanconformations in its crystal structures. Sharing the same S175G mutation with SCFP3A, Cerulean-S175G showed a slightly increased quantum yield, like SCFP3A, but did not adopt theECFPconformation adopted by SCFP3A. Detailed comparison of Cerulean-S175G and other ECFP variants revealed that the notable conformational changes in ECFP variants can be understood mainly in terms of the interaction between the Trp66 residue of the chromophore and residues 145–148 of β-strand 7.

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