International Journal of Molecular Sciences

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11 pages, 2047 KiB

Open AccessEditorial

Bioluminescent and Fluorescent Proteins: Molecular Mechanisms and Modern Applications

by Eugene S. Vysotski

Int. J. Mol. Sci. 2023, 24(1), 281; https://doi.org/10.3390/ijms24010281 - 23 Dec 2022

Cited by 2 | Viewed by 2263

Abstract

Light emission by living organisms in the visible spectrum range is called bioluminescence [...] Full article

(This article belongs to the Special Issue Bioluminescent and Fluorescent Proteins: Molecular Mechanisms and Modern Applications 3.0)

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Research

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15 pages, 1686 KiB

Open AccessArticle

Photodegradable by Yellow-Orange Light degFusionRed Optogenetic Module with Autocatalytically Formed Chromophore

by Konstantin G. Chernov, Kyrylo Yu. Manoilov, Olena S. Oliinyk, Daria M. Shcherbakova and Vladislav V. Verkhusha

Int. J. Mol. Sci. 2023, 24(7), 6526; https://doi.org/10.3390/ijms24076526 - 30 Mar 2023

Cited by 1 | Viewed by 2123

Abstract

Optogenetic systems driven by yellow-orange light are required for the simultaneous regulation of several cellular processes. We have engineered the red fluorescent protein FusionRed into a 26 kDa monomeric optogenetic module, called degFusionRed. Unlike other fluorescent protein-based optogenetic domains, which exhibit light-induced self-inactivation [...] Read more.

Optogenetic systems driven by yellow-orange light are required for the simultaneous regulation of several cellular processes. We have engineered the red fluorescent protein FusionRed into a 26 kDa monomeric optogenetic module, called degFusionRed. Unlike other fluorescent protein-based optogenetic domains, which exhibit light-induced self-inactivation by generating reactive oxygen species, degFusionRed undergoes proteasomal degradation upon illumination with 567 nm light. Similarly to the parent protein, degFusionRed has minimal absorbance at 450 nm and above 650 nm, making it spectrally compatible with blue and near-infrared-light-controlled optogenetic tools. The autocatalytically formed chromophore provides degFusionRed with an additional advantage over most optogenetic tools that require the binding of the exogenous chromophores, the amount of which varies in different cells. The degFusionRed efficiently performed in the engineered light-controlled transcription factor and in the targeted photodegradation of the protein of interest, demonstrating its versatility as the optogenetic module of choice for spectral multiplexed interrogation of various cellular processes. Full article

(This article belongs to the Special Issue Bioluminescent and Fluorescent Proteins: Molecular Mechanisms and Modern Applications 3.0)

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10 pages, 2315 KiB

Open AccessArticle

Crystal Structure of Bright Fluorescent Protein BrUSLEE with Subnanosecond Fluorescence Lifetime; Electric and Dynamic Properties

by Ekaterina Goryacheva, Roman Efremov, Nikolai Krylov, Igor Artemyev, Alexey Bogdanov, Anastasia Mamontova, Sergei Pletnev, Nadya Pletneva and Vladimir Pletnev

Int. J. Mol. Sci. 2023, 24(7), 6403; https://doi.org/10.3390/ijms24076403 - 29 Mar 2023

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Abstract

The rapid development of new microscopy techniques for cell biology has exposed the need for genetically encoded fluorescent tags with special properties. Fluorescent biomarkers of the same color and spectral range and different fluorescent lifetimes (FLs) became useful for fluorescent lifetime image microscopy [...] Read more.

The rapid development of new microscopy techniques for cell biology has exposed the need for genetically encoded fluorescent tags with special properties. Fluorescent biomarkers of the same color and spectral range and different fluorescent lifetimes (FLs) became useful for fluorescent lifetime image microscopy (FLIM). One such tag, the green fluorescent protein BrUSLEE (Bright Ultimately Short Lifetime Enhanced Emitter), having an extremely short subnanosecond component of fluorescence lifetime (FL~0.66 ns) and exceptional fluorescence brightness, was designed for FLIM experiments. Here, we present the X-ray structure and discuss the structure-functional relations of BrUSLEE. Its development from the EGFP (enhanced green fluorescent proteins) precursor (FL~2.83 ns) resulted in a change of the chromophore microenvironment due to a significant alteration in the side chain conformations. To get further insight into molecular details explaining the observed differences in the photophysical properties of these proteins, we studied their structural, dynamic, and electric properties by all-atom molecular-dynamics simulations in an aqueous solution. It has been shown that compared to BrUSLEE, the mobility of the chromophore in the EGFP is noticeably limited by nonbonded interactions (mainly H-bonds) with the neighboring residues. Full article

(This article belongs to the Special Issue Bioluminescent and Fluorescent Proteins: Molecular Mechanisms and Modern Applications 3.0)

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17 pages, 1308 KiB

Open AccessArticle

S-Series Coelenterazine-Driven Combinatorial Bioluminescence Imaging Systems for Mammalian Cells

by Genta Kamiya, Nobuo Kitada, Tadaomi Furuta, Takashi Hirano, Shojiro A. Maki and Sung-Bae Kim

Int. J. Mol. Sci. 2023, 24(2), 1420; https://doi.org/10.3390/ijms24021420 - 11 Jan 2023

Cited by 9 | Viewed by 3521

Abstract

A unique combinatorial bioluminescence (BL) imaging system was developed for determining molecular events in mammalian cells with various colors and BL intensity patterns. This imaging system consists of one or multiple reporter luciferases and a series of novel coelenterazine (CTZ) analogues named “S-series”. [...] Read more.

A unique combinatorial bioluminescence (BL) imaging system was developed for determining molecular events in mammalian cells with various colors and BL intensity patterns. This imaging system consists of one or multiple reporter luciferases and a series of novel coelenterazine (CTZ) analogues named “S-series”. For this study, ten kinds of novel S-series CTZ analogues were synthesized and characterized concerning the BL intensities, spectra, colors, and specificity of various marine luciferases. The characterization revealed that the S-series CTZ analogues luminesce with blue-to-orange-colored BL spectra with marine luciferases, where the most red-shifted BL spectrum peaked at 583 nm. The colors completed a visible light color palette with those of our precedent C-series CTZ analogues. The synthesized substrates S1, S5, S6, and S7 were found to have a unique specificity with marine luciferases, such as R86SG, NanoLuc (shortly, NLuc), and ALuc16. They collectively showed unique BL intensity patterns to identify the marine luciferases together with colors. The marine luciferases, R86SG, NLuc, and ALuc16, were multiplexed into multi-reporter systems, the signals of which were quantitatively unmixed with the specific substrates. When the utility was applied to a single-chain molecular strain probe, the imaging system simultaneously reported three different optical indexes for a ligand, i.e., unique BL intensity and color patterns for identifying the reporters, together with the ligand-specific fold intensities in mammalian cells. This study directs a new combinatorial BL imaging system to specific image molecular events in mammalian cells with multiple optical indexes. Full article

(This article belongs to the Special Issue Bioluminescent and Fluorescent Proteins: Molecular Mechanisms and Modern Applications 3.0)

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19 pages, 26447 KiB

Open AccessArticle

Direct or Indirect ESPT Mechanism in CFP psamFP488? A Theoretical-Computational Investigation

by Greta Donati and Nadia Rega

Int. J. Mol. Sci. 2022, 23(24), 15640; https://doi.org/10.3390/ijms232415640 - 9 Dec 2022

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Abstract

Fluorescent Proteins are widely studied for their multiple applications in technological and biotechnological fields. Despite this, they continue to represent a challenge in terms of a complete understanding of all the non-equilibrium photo-induced processes that rule their properties. In this context, a theoretical-computational [...] Read more.

Fluorescent Proteins are widely studied for their multiple applications in technological and biotechnological fields. Despite this, they continue to represent a challenge in terms of a complete understanding of all the non-equilibrium photo-induced processes that rule their properties. In this context, a theoretical-computational approach can support experimental results in unveiling and understanding the processes taking place after electronic excitation. A non-standard cyan fluorescent protein, psamFP488, is characterized by an absorption maximum that is blue-shifted in comparison to other cyan fluorescent proteins. This protein is characterized by an extended Stokes shift and an ultrafast (170 fs) excited state proton transfer. In this work, a theoretical-computational study, including excited state ab initio dynamics, is performed to help understanding the reaction mechanism and propose new hypotheses on the role of the residues surrounding the chromophore. Our results suggest that the proton transfer could be indirect toward the acceptor (Glu167) and involves other residues surrounding the chromophore, despite the ultrafast kinetics. Full article

(This article belongs to the Special Issue Bioluminescent and Fluorescent Proteins: Molecular Mechanisms and Modern Applications 3.0)

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21 pages, 6358 KiB

Open AccessArticle

cNTnC and fYTnC2, Genetically Encoded Green Calcium Indicators Based on Troponin C from Fast Animals

by Oksana M. Subach, Anna V. Vlaskina, Yuliya K. Agapova, Dmitriy A. Korzhenevskiy, Alena Y. Nikolaeva, Anna M. Varizhuk, Maksim F. Subach, Maxim V. Patrushev, Kiryl D. Piatkevich, Konstantin M. Boyko and Fedor V. Subach

Int. J. Mol. Sci. 2022, 23(23), 14614; https://doi.org/10.3390/ijms232314614 - 23 Nov 2022

Cited by 5 | Viewed by 1829

Abstract

NTnC-like green fluorescent genetically encoded calcium indicators (GECIs) with two calcium ion binding sites were constructed using the insertion of truncated troponin C (TnC) from Opsanus tau into green fluorescent proteins (GFPs). These GECIs are small proteins containing the N- and C-termini of [...] Read more.

NTnC-like green fluorescent genetically encoded calcium indicators (GECIs) with two calcium ion binding sites were constructed using the insertion of truncated troponin C (TnC) from Opsanus tau into green fluorescent proteins (GFPs). These GECIs are small proteins containing the N- and C-termini of GFP; they exert a limited effect on the cellular free calcium ion concentration; and in contrast to calmodulin-based calcium indicators they lack undesired interactions with intracellular proteins in neurons. The available TnC-based NTnC or YTnC GECIs had either an inverted response and high brightness but a limited dynamic range or a positive response and fast kinetics in neurons but lower brightness and an enhanced but still limited dF/F dynamic range. Here, we solved the crystal structure of NTnC at 2.5 Å resolution. Based on this structure, we developed positive NTnC2 and inverted iNTnC2 GECIs with a large dF/F dynamic range in vitro but very slow rise and decay kinetics in neurons. To overcome their slow responsiveness, we swapped TnC from O. tau in NTnC2 with truncated troponin C proteins from the muscles of fast animals, namely, the falcon, hummingbird, cheetah, bat, rattlesnake, and ant, and then optimized the resulting constructs using directed molecular evolution. Characterization of the engineered variants using purified proteins, mammalian cells, and neuronal cultures revealed cNTnC GECI with truncated TnC from Calypte anna (hummingbird) to have the largest dF/F fluorescence response and fast dissociation kinetics in neuronal cultures. In addition, based on the insertion of truncated TnCs from fast animals into YTnC2, we developed fYTnC2 GECI with TnC from Falco peregrinus (falcon). The purified proteins cNTnC and fYTnC2 had 8- and 6-fold higher molecular brightness and 7- and 6-fold larger dF/F responses to the increase in Ca²⁺ ion concentration than YTnC, respectively. cNTnC GECI was also 4-fold more photostable than YTnC and fYTnC2 GECIs. Finally, we assessed the developed GECIs in primary mouse neuronal cultures stimulated with an external electric field; in these conditions, cNTnC had a 2.4-fold higher dF/F fluorescence response than YTnC and fYTnC2 and was the same or slightly slower (1.4-fold) than fYTnC2 and YTnC in the rise and decay half-times, respectively. Full article

(This article belongs to the Special Issue Bioluminescent and Fluorescent Proteins: Molecular Mechanisms and Modern Applications 3.0)

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15 pages, 2163 KiB

Open AccessArticle

Investigation of the Anticancer and Drug Combination Potential of Brominated Coelenteramines toward Breast and Prostate Cancer

by Carla M. Magalhães, Patricia González-Berdullas, Mariana Pereira, Diana Duarte, Nuno Vale, Joaquim C. G. Esteves da Silva and Luís Pinto da Silva

Int. J. Mol. Sci. 2022, 23(22), 13981; https://doi.org/10.3390/ijms232213981 - 12 Nov 2022

Cited by 2 | Viewed by 1781

Abstract

Cancer is a very challenging disease to treat, both in terms of therapeutic efficiency and harmful side effects, which continues to motivate the pursuit for novel molecules with potential anticancer activity. Herein, we have designed, synthesized, and evaluated the cytotoxicity of different brominated [...] Read more.

Cancer is a very challenging disease to treat, both in terms of therapeutic efficiency and harmful side effects, which continues to motivate the pursuit for novel molecules with potential anticancer activity. Herein, we have designed, synthesized, and evaluated the cytotoxicity of different brominated coelenteramines, which are metabolic products and synthesis precursors of the chemi-/bioluminescent system of marine coelenterazine. The evaluation of the anticancer potential of these molecules was carried out for both prostate and breast cancer, while also exploring their potential for use in combination therapy. Our results provided further insight into the structure–activity relationship of this type of molecule, such as their high structural specificity, as well highlighting the 4-bromophenyl moiety as essential for the anticancer activity. The obtained data also indicated that, despite their similarity, the anticancer activity displayed by both brominated coelenteramines and coelenterazines should arise from independent mechanisms of action. Finally, one of the studied coelenteramines was able to improve the profile of a known chemotherapeutic agent, even at concentrations in which its anticancer activity was not relevant. Thus, our work showed the potential of different components of marine chemi-/bioluminescent systems as novel anticancer molecules, while providing useful information for future optimizations. Full article

(This article belongs to the Special Issue Bioluminescent and Fluorescent Proteins: Molecular Mechanisms and Modern Applications 3.0)

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14 pages, 2713 KiB

Open AccessArticle

C-Series Coelenterazine-Driven Bioluminescence Signature Imaging

by Genta Kamiya, Nobuo Kitada, Tadaomi Furuta, Takashi Hirano, Shojiro Maki and Sung Bae Kim

Int. J. Mol. Sci. 2022, 23(21), 13047; https://doi.org/10.3390/ijms232113047 - 27 Oct 2022

Cited by 7 | Viewed by 1839

Abstract

The present study introduces a unique BL signature imaging system with novel CTZ analogues named “C-series.” Nine kinds of C-series CTZ analogues were first synthesized, and BL intensity patterns and spectra were then examined according to the marine luciferases. The results show that [...] Read more.

The present study introduces a unique BL signature imaging system with novel CTZ analogues named “C-series.” Nine kinds of C-series CTZ analogues were first synthesized, and BL intensity patterns and spectra were then examined according to the marine luciferases. The results show that the four CTZ analogues named C3, C4, C6, and C7, individually or collectively luminesce with completely distinctive BL spectral signatures and intensity patterns according to the luciferases: Renilla luciferase (RLuc), NanoLuc, and artificial luciferase (ALuc). The signatural reporters were multiplexed into a multi-reporter system comprising RLuc8.6-535SG and ALuc16. The usefulness of the signatural reporters was further determined with a multi-probe system that consists of two single-chain probes embedding RLuc8 and ALuc23. This study is a great addition to the study of conventional bioassays with a unique methodology, and for the specification of each signal in a single- or multi-reporter system using unique BL signatures and patterns of reporter luciferases. Full article

(This article belongs to the Special Issue Bioluminescent and Fluorescent Proteins: Molecular Mechanisms and Modern Applications 3.0)

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