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

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>

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Genetically Encoded Photosensitizers as Light-Triggered Antimicrobial Agents

International Journal of Molecular Sciences ◽

10.3390/ijms20184608 ◽

2019 ◽

Vol 20 (18) ◽

pp. 4608 ◽

Cited By ~ 6

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.

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Utilizing Tyrosine Analogs to Alter Photophysical Properties of Green Fluorescent Protein

Biophysical Journal ◽

10.1016/j.bpj.2019.11.2615 ◽

2020 ◽

Vol 118 (3) ◽

pp. 471a

Author(s):

Darcy R. Harris ◽

Scott H. Brewer ◽

Christine M. Phillips-Piro

Keyword(s):

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Photophysical Properties ◽

Green Fluorescent

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A photostable monomeric superfolder GFP

10.1101/811588 ◽

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.

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Structural and spectrophotometric investigation of two unnatural amino-acid altered chromophores in the superfolder green fluorescent protein

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798321006525 ◽

2021 ◽

Vol 77 (8) ◽

Author(s):

Gregory M. Olenginski ◽

Juliana Piacentini ◽

Darcy R. Harris ◽

Nicolette A. Runko ◽

Brianna M. Papoutsis ◽

...

Keyword(s):

Amino Acids ◽

Green Fluorescent Protein ◽

Amino Acid ◽

Unnatural Amino Acids ◽

Fluorescent Protein ◽

Photophysical Properties ◽

Unnatural Amino Acid ◽

Green Fluorescent ◽

Absorbance Band ◽

Protein Constructs

The spectrophotometric properties of the green fluorescent protein (GFP) result from the post-translationally cyclized chromophore composed of three amino acids including a tyrosine at the center of the β-barrel protein. Altering the amino acids in the chromophore or the nearby region has resulted in numerous GFP variants with differing photophysical properties. To further examine the effect of small atomic changes in the chromophore on the structure and photophysical properties of GFP, the hydroxyl group of the chromophore tyrosine was replaced with a nitro or a cyano group. The structures and spectrophotometric properties of these superfolder GFP (sfGFP) variants with the unnatural amino acids (UAAs) 4-nitro-L-phenylalanine or 4-cyano-L-phenylalanine were explored. Notably, the characteristic 487 nm absorbance band of wild-type (wt) sfGFP is absent in both unnatural amino-acid-containing protein constructs (Tyr66pNO2Phe-sfGFP and Tyr66pCNPhe-sfGFP). Consequently, neither Tyr66pNO2Phe-sfGFP nor Tyr66pCNPhe-sfGFP exhibited the characteristic emission of wt sfGFP centered at 511 nm when excited at 487 nm. Tyr66pNO2Phe-sfGFP appeared orange due to an absorbance band centered at 406 nm that was not present in wt sfGFP, while Tyr66pCNPhe-sfGFP appeared colorless with an absorbance band centered at 365 nm. Mass spectrometry and X-ray crystallography confirmed the presence of a fully formed chromophore and no significant structural changes in either of these UAA-containing protein constructs, signaling that the change in the observed photophysical properties of the proteins is the result of the presence of the UAA in the chromophore.

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Five concomitant polymorphs of a green fluorescent protein chromophore (GFPc) analogue: understanding variations in photoluminescence with π-stacking interactions

Acta Crystallographica Section B Structural Science Crystal Engineering and Materials ◽

10.1107/s2052520620010343 ◽

2020 ◽

Vol 76 (5) ◽

pp. 850-864

Author(s):

Bhupendra P. Mali ◽

Soumya Ranjan Dash ◽

Shrikant B. Nikam ◽

Anisha Puthuvakkal ◽

Kumar Vanka ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Smart Materials ◽

Fluorescent Protein ◽

Photophysical Properties ◽

Carbonyl Oxygen ◽

Stacking Interactions ◽

Subtle Difference ◽

Sensing Applications ◽

Π Stacking ◽

Green Fluorescent

The synthetically modified green fluorescent protein chromophore analogue 3,4,5-trimethoxybenzylidene imidazolinone (1) yielded five polymorphs (I, II, III, IV, V) concomitantly irrespective of the solvent used for crystallization. The pentamorphic modification of 1 is solely due to the interplay of iso-energetic weak intermolecular interactions in molecular associations as well as the conformational flexibility offered by a C—C single bond, which connects the electron-deficient moiety imidazolinone with the electron-rich trimethoxybenzylidene group. A common structural feature observed in all the polymorphs is the formation of a `zero-dimensional' centrosymmetric dimeric unit through a short and linear C—H...O hydrogen bond engaging phenyl C—H and imidazolinone carbonyl oxygen. However, the networking of these dimeric units showed a subtle difference in all the polymorphs. The 2D isostructurality was observed between polymorphs I, II and III, while the other two polymorphs IV and V revealed only `zero-dimensional' isostructurality. The different fluorescence emissions of Form I (blue) and Forms II to V (yellow) were attributed to the differences in π-stacking interactions. It shows that one can modulate the photophysical properties of these smart materials by slightly altering their crystal structure. Such an approach will aid in developing new multi-colour organic fluorescent materials of varying crystal structures for live-cell imaging and fluorescent sensing applications.

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Plasmon-Enhanced Fluorescence of EGFP on Short-Range Ordered Ag Nanohole Arrays

Nanomaterials ◽

10.3390/nano10122563 ◽

2020 ◽

Vol 10 (12) ◽

pp. 2563

Author(s):

Vladimir E. Bochenkov ◽

Ekaterina M. Lobanova ◽

Aleksander M. Shakhov ◽

Artyom A. Astafiev ◽

Alexey M. Bogdanov ◽

...

Keyword(s):

Short Range ◽

Fluorescent Protein ◽

Fluorescent Proteins ◽

Photophysical Properties ◽

Plasmon Mode ◽

Plasmonic Nanostructures ◽

Enhanced Fluorescence ◽

Nanohole Arrays ◽

Covalently Linked ◽

Green Fluorescent

Fluorescence of organic molecules can be enhanced by plasmonic nanostructures through coupling to their locally amplified electromagnetic field, resulting in higher brightness and better photostability of fluorophores, which is particularly important for bioimaging applications involving fluorescent proteins as genetically encoded biomarkers. Here, we show that a hybrid bionanosystem comprised of a monolayer of Enhanced Green Fluorescent Protein (EGFP) covalently linked to optically thin Ag films with short-range ordered nanohole arrays can exhibit up to 6-fold increased brightness. The largest enhancement factor is observed for nanohole arrays with a propagating surface plasmon mode, tuned to overlap with both excitation and emission of EGFP. The fluorescence lifetime measurements in combination with FDTD simulations provide in-depth insight into the origin of the fluorescence enhancement, showing that the effect is due to the local amplification of the optical field near the edges of the nanoholes. Our results pave the way to improving the photophysical properties of hybrid bionanosystems based on fluorescent proteins at the interface with easily fabricated and tunable plasmonic nanostructures.

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