Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 42
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Adv Sci (Weinh) ; : e2102474, 2021 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-34533889

RESUMO

Photoacoustic tomography (PAT) with genetically encoded near-infrared probes enables visualization of specific cell populations in vivo at high resolution deeply in biological tissues. However, because of a lack of proper probes, PAT of cellular dynamics remains unexplored. Here, the authors report a near-infrared Forster resonance energy transfer (FRET) biosensor based on a miRFP670-iRFP720 pair of the near-infrared fluorescent proteins, which enables dynamic functional imaging of active biological processes in deep tissues. By photoacoustically detecting the changes in the optical absorption of the miRFP670 FRET-donor, they monitored cell apoptosis in deep tissue at high spatiotemporal resolution using PAT. Specifically, they detected apoptosis in single cells at a resolution of ≈3 µm in a mouse ear tumor, and in deep brain tumors (>3 mm beneath the scalp) of living mice at a spatial resolution of ≈150 µm with a 20 Hz frame rate. These results open the way for high-resolution photoacoustic imaging of dynamic biological processes in deep tissues using NIR biosensors and PAT.

2.
Nat Methods ; 18(9): 1027-1037, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34446923

RESUMO

Genetically encoded tools for the regulation of endogenous molecules (RNA, DNA elements and protein) are needed to study and control biological processes with minimal interference caused by protein overexpression and overactivation of signaling pathways. Here we focus on light-controlled optogenetic tools (OTs) that allow spatiotemporally precise regulation of gene expression and protein function. To control endogenous molecules, OTs combine light-sensing modules from natural photoreceptors with specific protein or nucleic acid binders. We discuss OT designs and group OTs according to the principles of their regulation. We outline characteristics of OT performance, discuss considerations for their use in vivo and review available OTs and their applications in cells and in vivo. Finally, we provide a brief outlook on the development of OTs.


Assuntos
Regulação da Expressão Gênica , Optogenética/métodos , Proteínas/metabolismo , Regulação Alostérica , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Luz , Mamíferos , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/metabolismo , Proteínas/química , Proteínas/genética
3.
J Neurosci Methods ; 362: 109314, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34375713

RESUMO

Genetically encoded fluorescent indicators of neuronal activity are ultimately developed to dissect functions of neuronal ensembles during behavior in living animals. Recent development of near-infrared shifted calcium and voltage indicators moved us closer to this goal and enabled crosstalk-free combination with blue light-controlled optogenetic tools for all-optical control and readout. Here I discuss designs of recent near-infrared and far-red calcium and voltage indicators, compare their properties and performance, and overview their applications to spectral multiplexing and in vivo imaging. I also provide perspectives for further development.


Assuntos
Cálcio , Optogenética , Animais , Corantes , Luz
4.
Methods Mol Biol ; 2350: 43-68, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34331278

RESUMO

Förster resonance energy transfer (FRET) biosensors are popular and useful for directly observing cellular signaling pathways in living cells. Until recently, multiplex imaging of genetically encoded FRET biosensors to simultaneously monitor several protein activities in one cell was limited due to a lack of spectrally compatible FRET pair of fluorescent proteins. With the recent development of miRFP series of near-infrared (NIR) fluorescent proteins, we are now able to extend the spectrum of FRET biosensors beyond blue-green-yellow into NIR. These new NIR FRET biosensors enable direct multiplex imaging together with commonly used cyan-yellow FRET biosensors. We describe herein a method to produce cell lines harboring two compatible FRET biosensors. We will then discuss how to directly multiplex-image these FRET biosensors in living cells. The approaches described herein are generally applicable to any combinations of genetically encoded, ratiometric FRET biosensors utilizing the cyan-yellow and NIR fluorescence.


Assuntos
Técnicas Biossensoriais/métodos , Imunofluorescência/métodos , Transdução de Sinais , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Proteínas de Transporte , Linhagem Celular , Ativação Enzimática , Transferência Ressonante de Energia de Fluorescência/métodos , Genes Reporter , Camundongos , Ligação Proteica , Proteínas rho de Ligação ao GTP/genética
5.
Nat Commun ; 12(1): 3859, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162879

RESUMO

Near-infrared (NIR) optogenetic systems for transcription regulation are in high demand because NIR light exhibits low phototoxicity, low scattering, and allows combining with probes of visible range. However, available NIR optogenetic systems consist of several protein components of large size and multidomain structure. Here, we engineer single-component NIR systems consisting of evolved photosensory core module of Idiomarina sp. bacterial phytochrome, named iLight, which are smaller and packable in adeno-associated virus. We characterize iLight in vitro and in gene transcription repression in bacterial and gene transcription activation in mammalian cells. Bacterial iLight system shows 115-fold repression of protein production. Comparing to multi-component NIR systems, mammalian iLight system exhibits higher activation of 65-fold in cells and faster 6-fold activation in deep tissues of mice. Neurons transduced with viral-encoded iLight system exhibit 50-fold induction of fluorescent reporter. NIR light-induced neuronal expression of green-light-activatable CheRiff channelrhodopsin causes 20-fold increase of photocurrent and demonstrates efficient spectral multiplexing.


Assuntos
Gammaproteobacteria/genética , Regulação da Expressão Gênica , Neurônios/metabolismo , Optogenética/métodos , Transcrição Genética/genética , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células Cultivadas , Feminino , Gammaproteobacteria/metabolismo , Células HeLa , Humanos , Raios Infravermelhos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Neurônios/citologia , Espectroscopia de Luz Próxima ao Infravermelho
6.
J Chem Phys ; 154(13): 135102, 2021 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-33832245

RESUMO

Reversibly photoswitchable probes allow for a wide variety of optical imaging applications. In particular, photoswitchable fluorescent probes have significantly facilitated the development of super-resolution microscopy. Recently, stimulated Raman scattering (SRS) imaging, a sensitive and chemical-specific optical microscopy, has proven to be a powerful live-cell imaging strategy. Driven by the advances of newly developed Raman probes, in particular the pre-resonance enhanced narrow-band vibrational probes, electronic pre-resonance SRS (epr-SRS) has achieved super-multiplex imaging with sensitivity down to 250 nM and multiplexity up to 24 colors. However, despite the high demand, photoswitchable Raman probes have yet to be developed. Here, we propose a general strategy for devising photoswitchable epr-SRS probes. Toward this goal, we exploit the molecular electronic and vibrational coupling, in which we switch the electronic states of the molecules to four different states to turn their ground-state epr-SRS signals on and off. First, we showed that inducing transitions to both the electronic excited state and triplet state can effectively diminish the SRS peaks. Second, we revealed that the epr-SRS signals can be effectively switched off in red-absorbing organic molecules through light-facilitated transitions to a reduced state. Third, we identified that photoswitchable proteins with near-infrared photoswitchable absorbance, whose states are modulable with their electronic resonances detunable toward and away from the pump photon energy, can function as the photoswitchable epr-SRS probes with desirable sensitivity (<1 µM) and low photofatigue (>40 cycles). These photophysical characterizations and proof-of-concept demonstrations should advance the development of novel photoswitchable Raman probes and open up the unexplored Raman imaging capabilities.

7.
Nat Biotechnol ; 39(3): 368-377, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33106681

RESUMO

While calcium imaging has become a mainstay of modern neuroscience, the spectral properties of current fluorescent calcium indicators limit deep-tissue imaging as well as simultaneous use with other probes. Using two monomeric near-infrared (NIR) fluorescent proteins (FPs), we engineered an NIR Förster resonance energy transfer (FRET)-based genetically encoded calcium indicator (iGECI). iGECI exhibits high levels of brightness and photostability and an increase up to 600% in the fluorescence response to calcium. In dissociated neurons, iGECI detects spontaneous neuronal activity and electrically and optogenetically induced firing. We validated the performance of iGECI up to a depth of almost 400 µm in acute brain slices using one-photon light-sheet imaging. Applying hybrid photoacoustic and fluorescence microscopy, we simultaneously monitored neuronal and hemodynamic activities in the mouse brain through an intact skull, with resolutions of ~3 µm (lateral) and ~25-50 µm (axial). Using two-photon imaging, we detected evoked and spontaneous neuronal activity in the mouse visual cortex, with fluorescence changes of up to 25%. iGECI allows biosensors and optogenetic actuators to be multiplexed without spectral crosstalk.


Assuntos
Cálcio/química , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Animais , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Humanos , Camundongos , Neurônios/fisiologia , Córtex Visual/diagnóstico por imagem , Córtex Visual/fisiologia
8.
ACS Chem Neurosci ; 11(21): 3523-3531, 2020 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-33063984

RESUMO

We developed genetically encoded voltage indicators using a transmembrane voltage-sensing domain and bright near-infrared fluorescent proteins derived from bacterial phytochromes. These new voltage indicators are excited by 640 nm light and emission is measured at 670 nm, allowing imaging in the near-infrared tissue transparency window. The spectral properties of our new indicators permit seamless voltage imaging with simultaneous blue-green light optogenetic actuator activation as well as simultaneous voltage-calcium imaging when paired with green calcium indicators. Iterative optimizations led to a fluorescent probe, here termed nirButterfly, which reliably reports neuronal activities including subthreshold membrane potential depolarization and hyperpolarization as well as spontaneous spiking or electrically- and optogenetically evoked action potentials. This enables largely improved all-optical causal interrogations of physiology.


Assuntos
Neurônios , Optogenética , Potenciais de Ação , Corantes Fluorescentes , Proteínas Luminescentes/genética , Proteínas
9.
Nat Commun ; 11(1): 239, 2020 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-31932632

RESUMO

Bright monomeric near-infrared (NIR) fluorescent proteins (FPs) are in high demand as protein tags for multicolor microscopy and in vivo imaging. Here we apply rational design to engineer a complete set of monomeric NIR FPs, which are the brightest genetically encoded NIR probes. We demonstrate that the enhanced miRFP series of NIR FPs, which combine high effective brightness in mammalian cells and monomeric state, perform well in both nanometer-scale imaging with diffraction unlimited stimulated emission depletion (STED) microscopy and centimeter-scale imaging in mice. In STED we achieve ~40 nm resolution in live cells. In living mice we detect ~105 fluorescent cells in deep tissues. Using spectrally distinct monomeric NIR FP variants, we perform two-color live-cell STED microscopy and two-color imaging in vivo. Having emission peaks from 670 nm to 720 nm, the next generation of miRFPs should become versatile NIR probes for multiplexed imaging across spatial scales in different modalities.


Assuntos
Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Imagem Molecular/instrumentação , Animais , Linhagem Celular , Feminino , Fluorescência , Humanos , Microscopia Intravital , Camundongos , Imagem Molecular/métodos , Engenharia de Proteínas , Estabilidade Proteica , Espectroscopia de Luz Próxima ao Infravermelho
10.
Front Cell Neurosci ; 13: 474, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31708747

RESUMO

Understanding how neuronal activity patterns in the brain correlate with complex behavior is one of the primary goals of modern neuroscience. Chemical transmission is the major way of communication between neurons, however, traditional methods of detection of neurotransmitter and neuromodulator transients in mammalian brain lack spatiotemporal precision. Modern fluorescent biosensors for neurotransmitters and neuromodulators allow monitoring chemical transmission in vivo with millisecond precision and single cell resolution. Changes in the fluorescent biosensor brightness occur upon neurotransmitter binding and can be detected using fiber photometry, stationary microscopy and miniaturized head-mounted microscopes. Biosensors can be expressed in the animal brain using adeno-associated viral vectors, and their cell-specific expression can be achieved with Cre-recombinase expressing animals. Although initially fluorescent biosensors for chemical transmission were represented by glutamate biosensors, nowadays biosensors for GABA, acetylcholine, glycine, norepinephrine, and dopamine are available as well. In this review, we overview functioning principles of existing intensiometric and ratiometric biosensors and provide brief insight into the variety of neurotransmitter-binding proteins from bacteria, plants, and eukaryotes including G-protein coupled receptors, which may serve as neurotransmitter-binding scaffolds. We next describe a workflow for development of neurotransmitter and neuromodulator biosensors. We then discuss advanced setups for functional imaging of neurotransmitter transients in the brain of awake freely moving animals. We conclude by providing application examples of biosensors for the studies of complex behavior with the single-neuron precision.

11.
Sci Rep ; 9(1): 1866, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30755663

RESUMO

Phytochromes are red/far-red light sensing photoreceptors employing linear tetrapyrroles as chromophores, which are covalently bound to a cysteine (Cys) residue in the chromophore-binding domain (CBD, composed of a PAS and a GAF domain). Recently, near-infrared (NIR) fluorescent proteins (FPs) engineered from bacterial phytochromes binding biliverdin IXα (BV), such as the iRFP series, have become invaluable probes for multicolor fluorescence microscopy and in vivo imaging. However, all current NIR FPs suffer from relatively low brightness. Here, by combining biochemical, spectroscopic and resonance Raman (RR) assays, we purified and characterized an iRFP variant that contains a BV chromophore simultaneously bound to two cysteines. This protein with the unusual double-Cys attached BV showed the highest fluorescence quantum yield (FQY) of 16.6% reported for NIR FPs, whereas the initial iRFP appeared to be a mixture of species with a mean FQY of 11.1%. The purified protein was also characterized with 1.3-fold higher extinction coefficient that together with FQY resulted in almost two-fold brighter fluorescence than the original iRFP as isolated. This work shows that the high FQY of iRFPs with two cysteines is a direct consequence of the double attachment. The PAS-Cys, GAF-Cys and double-Cys attachment each entails distinct configurational constraints of the BV adduct, which can be identified by distinct RR spectroscopic features, i.e. the marker band including the C=C stretching coordinate of the ring A-B methine bridge, which was previously identified as being characteristic for rigid chromophore embedment and high FQY. Our findings can be used to rationally engineer iRFP variants with enhanced FQYs.


Assuntos
Cisteína/química , Proteínas Luminescentes/química , Proteínas de Bactérias/química , Biliverdina/química , Escherichia coli/química , Mutagênese , Fitocromo/química , Ligação Proteica , Domínios Proteicos , Rodopseudomonas/química , Espectrofotometria Ultravioleta , Análise Espectral Raman , Zinco/química
12.
Nat Commun ; 10(1): 279, 2019 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-30655515

RESUMO

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.


Assuntos
Proteínas de Bactérias/química , Técnicas Biossensoriais/métodos , Cianobactérias/química , Proteínas Luminescentes/química , Fotorreceptores Microbianos/química , Células 3T3 , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Biliverdina/metabolismo , Cianobactérias/genética , Cianobactérias/metabolismo , Evolução Molecular Direcionada/métodos , Feminino , Fluorescência , Células HeLa , Humanos , Microscopia Intravital/métodos , Proteínas Luminescentes/genética , Proteínas Luminescentes/isolamento & purificação , Proteínas Luminescentes/metabolismo , Camundongos , Microscopia de Fluorescência/métodos , Mutagênese , Neurônios , Optogenética/métodos , Fotorreceptores Microbianos/genética , Fotorreceptores Microbianos/isolamento & purificação , Fotorreceptores Microbianos/metabolismo , Cultura Primária de Células , Domínios Proteicos/genética , Engenharia de Proteínas , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Espectroscopia de Luz Próxima ao Infravermelho/métodos
13.
Nat Commun ; 9(1): 2734, 2018 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-30013153

RESUMO

Photoacoustic (PA) computed tomography (PACT) benefits from genetically encoded probes with photochromic behavior, which dramatically increase detection sensitivity and specificity through photoswitching and differential imaging. Starting with a DrBphP bacterial phytochrome, we have engineered a near-infrared photochromic probe, DrBphP-PCM, which is superior to the full-length RpBphP1 phytochrome previously used in differential PACT. DrBphP-PCM has a smaller size, better folding, and higher photoswitching contrast. We have imaged both DrBphP-PCM and RpBphP1 simultaneously on the basis of their unique signal decay characteristics, using a reversibly switchable single-impulse panoramic PACT (RS-SIP-PACT) with a single wavelength excitation. The simple structural organization of DrBphP-PCM allows engineering a bimolecular PA complementation reporter, a split version of DrBphP-PCM, termed DrSplit. DrSplit enables PA detection of protein-protein interactions in deep-seated mouse tumors and livers, achieving 125-µm spatial resolution and 530-cell sensitivity in vivo. The combination of RS-SIP-PACT with DrBphP-PCM and DrSplit holds great potential for noninvasive multi-contrast deep-tissue functional imaging.


Assuntos
Proteínas de Bactérias/genética , Neoplasias Encefálicas/diagnóstico por imagem , Fígado/diagnóstico por imagem , Imagem Molecular/métodos , Técnicas Fotoacústicas/métodos , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Tomografia/métodos , Animais , Proteínas de Bactérias/metabolismo , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Deinococcus/genética , Deinococcus/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Feminino , Expressão Gênica , Células HEK293 , Xenoenxertos , Humanos , Fígado/metabolismo , Camundongos , Camundongos Nus , Imagem Molecular/instrumentação , Técnicas Fotoacústicas/instrumentação , Plasmídeos/química , Plasmídeos/metabolismo , Engenharia de Proteínas , Mapeamento de Interação de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rodopseudomonas/genética , Rodopseudomonas/metabolismo , Espectroscopia de Luz Próxima ao Infravermelho/instrumentação , Tomografia/instrumentação
14.
Trends Biotechnol ; 36(12): 1230-1243, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30041828

RESUMO

Since mammalian tissue is relatively transparent to near-infrared (NIR) light, NIR fluorescent proteins (FPs) engineered from bacterial phytochromes have become widely used probes for non-invasive in vivo imaging. Recently, these genetically encoded NIR probes have been substantially improved, enabling imaging experiments that were not possible previously. Here, we discuss the use of monomeric NIR FPs and NIR biosensors for multiplexed imaging with common visible GFP-based probes and blue light-activatable optogenetic tools. These NIR probes are suitable for visualization of functional activities from molecular to organismal levels. In combination with advanced imaging techniques, such as two-photon microscopy with adaptive optics, photoacoustic tomography and its recent modification reversibly switchable photoacoustic computed tomography, NIR probes allow subcellular resolution at millimeter depths.


Assuntos
Microscopia Intravital/métodos , Proteínas Luminescentes/análise , Imagem Óptica/métodos , Optogenética/métodos , Proteínas Luminescentes/genética
15.
Sci Rep ; 8(1): 11123, 2018 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-30042404

RESUMO

A conventional photoacoustic microscopy (PAM) system typically has to make tradeoffs between its spatial resolution and penetration depth, by choosing a fixed configuration of optical excitation and acoustic detection. The single-scale imaging capability of PAM may limit its applications in biomedical studies. Here, we report a quad-mode photoacoustic microscopy (QM-PAM) system with four complementary spatial resolutions and maximum penetration depths. For this we first developed a ring-shaped focused ultrasound transducer that has two independent elements with respective central frequencies at 20 MHz and 40 MHz, providing complementary acoustically-determined spatial resolutions and penetration depths. To accommodate the dual-element ultrasound transducer, we implemented two optical excitation modes to provide tightly- and weakly-focused light illumination. The dual-element acoustic detection combined with the two optical focusing modes can thus provide four imaging scales in a single imaging device, with consistent contrast mechanisms and co-registered field of views. We have demonstrated the multiscale morphological, functional, and molecular imaging capability of QM-PAM in the mouse head, leg and ear in vivo. We expect the high scale flexibility of QM-PAM will enable broad applications in preclinical studies.


Assuntos
Cabeça/diagnóstico por imagem , Técnicas Fotoacústicas/métodos , Ultrassonografia/métodos , Animais , Cabeça/fisiologia , Camundongos , Microscopia , Análise Espectral/métodos , Transdutores
16.
Nat Chem Biol ; 14(9): 902, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29728601

RESUMO

In the version of this article originally published, the values for time shown on the x axis of Figure 5c were incorrect. The error has been corrected in all versions of the paper.

17.
Nat Chem Biol ; 14(6): 591-600, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29686359

RESUMO

Direct visualization and light control of several cellular processes is a challenge, owing to the spectral overlap of available genetically encoded probes. Here we report the most red-shifted monomeric near-infrared (NIR) fluorescent protein, miRFP720, and the fully NIR Förster resonance energy transfer (FRET) pair miRFP670-miRFP720, which together enabled design of biosensors compatible with CFP-YFP imaging and blue-green optogenetic tools. We developed a NIR biosensor for Rac1 GTPase and demonstrated its use in multiplexed imaging and light control of Rho GTPase signaling pathways. Specifically, we combined the Rac1 biosensor with CFP-YFP FRET biosensors for RhoA and for Rac1-GDI binding, and concurrently used the LOV-TRAP tool for upstream Rac1 activation. We directly observed and quantified antagonism between RhoA and Rac1 dependent on the RhoA-downstream effector ROCK; showed that Rac1 activity and GDI binding closely depend on the spatiotemporal coordination between these two molecules; and simultaneously observed Rac1 activity during optogenetic manipulation of Rac1.


Assuntos
Transferência Ressonante de Energia de Fluorescência , Proteínas rho de Ligação ao GTP/química , Animais , Técnicas Biossensoriais , Bradyrhizobium , Células HEK293 , Células HeLa , Humanos , Camundongos , Células NIH 3T3 , Optogenética , Plasmídeos , Ligação Proteica , Transdução de Sinais , Espectroscopia de Luz Próxima ao Infravermelho , Proteína cdc42 de Ligação ao GTP/química , Proteínas rac1 de Ligação ao GTP/química
18.
Chem Sci ; 8(6): 4546-4557, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28936332

RESUMO

Brighter near-infrared (NIR) fluorescent proteins (FPs) are required for multicolor microscopy and deep-tissue imaging. Here, we present structural and biochemical analyses of three monomeric, spectrally distinct phytochrome-based NIR FPs, termed miRFPs. The miRFPs are closely related and differ by only a few amino acids, which define their molecular brightness, brightness in mammalian cells, and spectral properties. We have identified the residues responsible for the spectral red-shift, revealed a new chromophore bound simultaneously to two cysteine residues in the PAS and GAF domains in blue-shifted NIR FPs, and uncovered the importance of amino acid residues in the N-terminus of NIR FPs for their molecular and cellular brightness. The novel chromophore covalently links the N-terminus of NIR FPs with their C-terminal GAF domain, forming a topologically closed knot in the structure, and also contributes to the increased brightness. Based on our studies, we suggest a strategy to develop spectrally distinct NIR FPs with enhanced brightness.

19.
Int J Mol Sci ; 18(5)2017 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-28481303

RESUMO

Near-infrared (NIR) fluorescent proteins (FPs) designed from PAS (Per-ARNT-Sim repeats) and GAF (cGMP phosphodiesterase/adenylate cyclase/FhlA transcriptional activator) domains of bacterial phytochromes covalently bind biliverdin (BV) chromophore via one or two Cys residues. We studied BV interaction with a series of NIR FP variants derived from the recently reported BphP1-FP protein. The latter was engineered from a bacterial phytochrome RpBphP1, and has two reactive Cys residues (Cys15 in the PAS domain and Cys256 in the GAF domain), whereas its mutants contain single Cys residues either in the PAS domain or in the GAF domain, or no Cys residues. We characterized BphP1-FP and its mutants biochemically and spectroscopically in the absence and in the presence of denaturant. We found that all BphP1-FP variants are monomers. We revealed that spectral properties of the BphP1-FP variants containing either Cys15 or Cys256, or both, are determined by the covalently bound BV chromophore only. Consequently, this suggests an involvement of the inter-monomeric allosteric effects in the BV interaction with monomers in dimeric NIR FPs, such as iRFPs. Likely, insertion of the Cys15 residue, in addition to the Cys256 residue, in dimeric NIR FPs influences BV binding by promoting the BV chromophore covalent cross-linking to both PAS and GAF domains.


Assuntos
Proteínas de Bactérias/metabolismo , Biliverdina/metabolismo , Proteínas Luminescentes/metabolismo , Fitocromo/metabolismo , Regulação Alostérica , Substituição de Aminoácidos , Proteínas de Bactérias/química , Biliverdina/química , Sítios de Ligação , Cisteína/genética , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Fitocromo/química , Fitocromo/genética , Ligação Proteica , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
20.
Sci Rep ; 6: 37362, 2016 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-27857208

RESUMO

Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes (BphPs) are of great interest for in vivo imaging. They utilize biliverdin (BV) as a chromophore, which is a heme degradation product, and therefore they are straightforward to use in mammalian tissues. Here, we report on fluorescence properties of NIR FPs with key alterations in their BV binding sites. BphP1-FP, iRFP670 and iRFP682 have Cys residues in both PAS and GAF domains, rather than in the PAS domain alone as in wild-type BphPs. We found that NIR FP variants with Cys in the GAF or with Cys in both PAS and GAF show blue-shifted emission with long fluorescence lifetimes. In contrast, mutants with Cys in the PAS only or no Cys residues at all exhibit red-shifted emission with shorter lifetimes. Combining these results with previous biochemical and BphP1-FP structural data, we conclude that BV adducts bound to Cys in the GAF are the origin of bright blue-shifted fluorescence. We propose that the long fluorescence lifetime follows from (i) a sterically more constrained thioether linkage, leaving less mobility for ring A than in canonical BphPs, and (ii) that π-electron conjugation does not extend on ring A, making excited-state deactivation less sensitive to ring A mobility.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas Luminescentes/metabolismo , Fitocromo/metabolismo , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Biliverdina/química , Sítios de Ligação/genética , Fluorescência , Proteínas Luminescentes/química , Proteínas Luminescentes/genética , Mutação , Fitocromo/genética , Homologia de Sequência de Aminoácidos , Espectrometria de Fluorescência , Espectroscopia de Luz Próxima ao Infravermelho
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...