Bacterial Phytochromes, Cyanobacteriochromes and Allophycocyanins as a Source of Near-Infrared Fluorescent Probes

Bacterial photoreceptors absorb light energy and transform it into intracellular signals that regulate metabolism. Bacterial phytochrome photoreceptors (BphPs), some cyanobacteriochromes (CBCRs) and allophycocyanins (APCs) possess the near-infrared (NIR) absorbance spectra that make them promising molecular templates to design NIR fluorescent proteins (FPs) and biosensors for studies in mammalian cells and whole animals. Here, we review structures, photochemical properties and molecular functions of several families of bacterial photoreceptors. We next analyze molecular evolution approaches to develop NIR FPs and biosensors. We then discuss phenotypes of current BphP-based NIR FPs and compare them with FPs derived from CBCRs and APCs. Lastly, we overview imaging applications of NIR FPs in live cells and in vivo. Our review provides guidelines for selection of existing NIR FPs, as well as engineering approaches to develop NIR FPs from the novel natural templates such as CBCRs.Peer reviewe

Smallest near-infrared fluorescent protein evolved from cyanobacteriochrome as versatile tag for spectral multiplexing

From a single domain of cyanobacteriochrome (CBCR) we developed a near-infrared (NIR) fluorescent protein (FP), termed miRFP670nano, with excitation at 645 nm and emission at 670 nm. This is the first CBCR-derived NIR FP evolved to efficiently bind endogenous biliverdin chromophore and brightly fluoresce in mammalian cells. miRFP670nano is a monomer with molecular weight of 17 kDa that is 2-fold smaller than bacterial phytochrome (BphP)-based NIR FPs and 1.6-fold smaller than GFP-like FPs. Crystal structure of the CBCR-based NIR FP with biliverdin reveals a molecular basis of its spectral and biochemical properties. Unlike BphP-derived NIR FPs, miRFP670nano is highly stable to denaturation and degradation and can be used as an internal protein tag. miRFP670nano is an effective FRET donor for red-shifted NIR FPs, enabling engineering NIR FRET biosensors spectrally compatible with GFP-like FPs and blue-green optogenetic tools. miRFP670nano unlocks a new source of diverse CBCR templates for NIR FPs.Peer reviewe

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

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

Single-domain near-infrared protein provides a scaffold for antigen-dependent fluorescent nanobodies

miRFP670nano3 offers improved near-infrared imaging and was used to develop fluorescent nanobodies whose stability and fluorescence strongly depend on antigen binding, with broad implications for detecting and manipulating cellular targets. Small near-infrared (NIR) fluorescent proteins (FPs) are much needed as protein tags for imaging applications. We developed a 17 kDa NIR FP, called miRFP670nano3, which brightly fluoresces in mammalian cells and enables deep-brain imaging. By exploring miRFP670nano3 as an internal tag, we engineered 32 kDa NIR fluorescent nanobodies, termed NIR-Fbs, whose stability and fluorescence strongly depend on the presence of specific intracellular antigens. NIR-Fbs allowed background-free visualization of endogenous proteins, detection of viral antigens, labeling of cells expressing target molecules and identification of double-positive cell populations with bispecific NIR-Fbs against two antigens. Applying NIR-Fbs as destabilizing fusion partners, we developed molecular tools for directed degradation of targeted proteins, controllable protein expression and modulation of enzymatic activities. Altogether, NIR-Fbs enable the detection and manipulation of a variety of cellular processes based on the intracellular protein profile.Peer reviewe

Cтратегии и технологии инновационного развития корпораций

У монографії представлено результати дослідження й систематизації теоретичних, науково-методологічних і практичних положень та розробок щодо стратегій та технологій інноваційного розвитку корпорацій. Запропоновано і обґрунтовано технології управління інноваційним розвитком підприємств, стратегії розвитку бізнесу, визначено сучасні реалії та тенденції корпоративного маркетингу, культури та соціальної відповідальності бізнесу, запропоновано нові підходи у корпоративному управлінні, обґрунтовано доцільність використання краудтехнологій фінансування інноваційних проектів, визначено підхо-ди щодо управління персоналом корпорацій в поведінковій економіці. Для науковців та фахівців сфери економіки та управління підприємствам

Development of bright red-shifted miRFP704nano using structural analysis of miRFPnano proteins

Abstract We recently converted the GAF domain of NpR3784 cyanobacteriochrome into near-infrared (NIR) fluorescent proteins (FPs). Unlike cyanobacterichrome, which incorporates phycocyanobilin tetrapyrrole, engineered NIR FPs bind biliverdin abundant in mammalian cells, thus being the smallest scaffold for it. Here, we determined the crystal structure of the brightest blue-shifted protein of the series, miRFP670nano3, at 1.8 Å resolution, characterized its chromophore environment and explained the molecular basis of its spectral properties. Using the determined structure, we have rationally designed a red-shifted NIR FP, termed miRFP704nano, with excitation at 680?nm and emission at 704?nm. miRFP704nano exhibits a small size of 17 kDa, enhanced molecular brightness, photostability and pH-stability. miRFP704nano performs well in various protein fusions in live mammalian cells and should become a versatile genetically-encoded NIR probe for multiplexed imaging across spatial scales in different modalities. This article is protected by copyright. All rights reserved.Peer reviewe

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

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

Blue-shift photoconversion of near-infrared fluorescent proteins for labeling and tracking in living cells and organisms

Abstract Photolabeling of intracellular molecules is an invaluable approach to studying various dynamic processes in living cells with high spatiotemporal precision. Among fluorescent proteins, photoconvertible mechanisms and their products are in the visible spectrum (400–650 nm), limiting their in vivo and multiplexed applications. Here we report the phenomenon of near-infrared to far-red photoconversion in the miRFP family of near infrared fluorescent proteins engineered from bacterial phytochromes. This photoconversion is induced by near-infrared light through a non-linear process, further allowing optical sectioning. Photoconverted miRFP species emit fluorescence at 650 nm enabling photolabeling entirely performed in the near-infrared range. We use miRFPs as photoconvertible fluorescent probes to track organelles in live cells and in vivo, both with conventional and super-resolution microscopy. The spectral properties of miRFPs complement those of GFP-like photoconvertible proteins, allowing strategies for photoconversion and spectral multiplexed applications

Blue-shift photoconversion of near-infrared fluorescent proteins for labeling and tracking in living cells and organisms

Photolabeling of intracellular molecules is an invaluable approach to studying various dynamic processes in living cells with high spatiotemporal precision. Among fluorescent proteins, photoconvertible mechanisms and their products are in the visible spectrum (400–650 nm), limiting their in vivo and multiplexed applications. Here we report the phenomenon of near-infrared to far-red photoconversion in the miRFP family of near infrared fluorescent proteins engineered from bacterial phytochromes. This photoconversion is induced by near-infrared light through a non-linear process, further allowing optical sectioning. Photoconverted miRFP species emit fluorescence at 650 nm enabling photolabeling entirely performed in the near-infrared range. We use miRFPs as photoconvertible fluorescent probes to track organelles in live cells and in vivo, both with conventional and super-resolution microscopy. The spectral properties of miRFPs complement those of GFP-like photoconvertible proteins, allowing strategies for photoconversion and spectral multiplexed applications.Peer reviewe