Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 144
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
J Mol Biol ; : 167359, 2021 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-34798132

RESUMO

Biliverdin-binding serpins (BBSs) are proteins that are responsible for coloration in amphibians and fluoresce in the near-infrared (NIR) spectral region. Here we produced the first functional recombinant BBS of the polka-dot treefrog Boana punctata (BpBBS), assembled with its biliverdin (BV) chromophore, and report its biochemical and photochemical characterization. We determined the crystal structure of BpBBS at 2.05 Å resolution, which demonstrated its structural homology to the mammalian protease inhibitor alpha-1-antitrypsin. BV interaction with BpBBS was studied and it was found that the N-terminal polypeptide (residues 19-50) plays a critical role in the BV binding. By comparing BpBBS with the available NIR fluorescent proteins and expressing it in mammalian cells, we demonstrated its potential as a NIR imaging probe. These results provide insight into the non-inhibitory function of serpins, provide a basis for improving their performance in mammalian cells, and suggest possible paths for the development of BBS-based fluorescent probes.

2.
Commun Chem ; 42021.
Artigo em Inglês | MEDLINE | ID: mdl-34746444

RESUMO

Near-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes are widely used for structural and functional deep-tissue imaging in vivo. To fluoresce, NIR FPs covalently bind a chromophore, such as biliverdin IXa tetrapyrrole. The efficiency of biliverdin binding directly affects the fluorescence properties, rendering understanding of its molecular mechanism of major importance. miRFP proteins constitute a family of bright monomeric NIR FPs that comprise a Per-ARNT-Sim (PAS) and cGMP-specific phosphodiesterases - Adenylyl cyclases - FhlA (GAF) domain. Here, we structurally analyze biliverdin binding to miRFPs in real time using time-resolved stimulated Raman spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations. Biliverdin undergoes isomerization, localization to its binding pocket, and pyrrolenine nitrogen protonation in <1 min, followed by hydrogen bond rearrangement in ~2 min. The covalent attachment to a cysteine in the GAF domain was detected in 4.3 min and 19 min in miRFP670 and its C20A mutant, respectively. In miRFP670, a second C-S covalent bond formation to a cysteine in the PAS domain occurred in 14 min, providing a rigid tetrapyrrole structure with high brightness. Our findings provide insights for the rational design of NIR FPs and a novel method to assess cofactor binding to light-sensitive proteins.

3.
Adv Sci (Weinh) ; 8(21): e2102474, 2021 11.
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.

4.
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
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.
Chem Sci ; 11(37): 10019-10034, 2020 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-33209247

RESUMO

Inhibition of receptor tyrosine kinases (RTKs) by small molecule inhibitors and monoclonal antibodies is used to treat cancer. Conversely, activation of RTKs with their ligands, including growth factors and insulin, is used to treat diabetes and neurodegeneration. However, conventional therapies that rely on injection of RTK inhibitors or activators do not provide spatiotemporal control over RTK signaling, which results in diminished efficiency and side effects. Recently, a number of optogenetic and optochemical approaches have been developed that allow RTK inhibition or activation in cells and in vivo with light. Light irradiation can control RTK signaling non-invasively, in a dosed manner, with high spatio-temporal precision, and without the side effects of conventional treatments. Here we provide an update on the current state of the art of optogenetic and optochemical RTK technologies and the prospects of their use in translational studies and therapy.

9.
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
10.
J Mol Biol ; 432(13): 3749-3760, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32302608

RESUMO

Optically controlled receptor tyrosine kinases (opto-RTKs) allow regulation of RTK signaling using light. Until recently, the majority of opto-RTKs were activated with blue-green light. Fusing a photosensory core module of Deinococcus radiodurans bacterial phytochrome (DrBphP-PCM) to the kinase domains of neurotrophin receptors resulted in opto-RTKs controlled with light above 650 nm. To expand this engineering approach to RTKs of other families, here we combined the DrBpP-PCM with the cytoplasmic domains of EGFR and FGFR1. The resultant Dr-EGFR and Dr-FGFR1 opto-RTKs are rapidly activated with near-infrared and inactivated with far-red light. The opto-RTKs efficiently trigger ERK1/2, PI3K/Akt, and PLCγ signaling. Absence of spectral crosstalk between the opto-RTKs and green fluorescent protein-based biosensors enables simultaneous Dr-FGFR1 activation and detection of calcium transients. Action mechanism of the DrBphP-PCM-based opto-RTKs is considered using the available RTK structures. DrBphP-PCM represents a versatile scaffold for engineering of opto-RTKs that are reversibly regulated with far-red and near-infrared light.


Assuntos
Fitocromo/ultraestrutura , Receptores Proteína Tirosina Quinases/ultraestrutura , Engenharia Tecidual/métodos , Tecidos Suporte/química , Técnicas Biossensoriais , Deinococcus/química , Deinococcus/genética , Proteínas de Fluorescência Verde/química , Células HeLa , Humanos , Luz , Sistema de Sinalização das MAP Quinases/efeitos da radiação , Fatores de Crescimento Neural/química , Fatores de Crescimento Neural/genética , Fosfatidilinositol 3-Quinases/genética , Fitocromo/química , Fitocromo/genética , Conformação Proteica/efeitos da radiação , Proteínas Proto-Oncogênicas c-akt/genética , Receptores Proteína Tirosina Quinases/química , Receptores Proteína Tirosina Quinases/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/química , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptores de Fator de Crescimento Neural/química , Receptores de Fator de Crescimento Neural/genética , Transdução de Sinais/efeitos da radiação
11.
Nat Commun ; 11(1): 605, 2020 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-32001718

RESUMO

Techniques of protein regulation, such as conditional gene expression, RNA interference, knock-in and knock-out, lack sufficient spatiotemporal accuracy, while optogenetic tools suffer from non-physiological response due to overexpression artifacts. Here we present a near-infrared light-activatable optogenetic system, which combines the specificity and orthogonality of intrabodies with the spatiotemporal precision of optogenetics. We engineer optically-controlled intrabodies to regulate genomically expressed protein targets and validate the possibility to further multiplex protein regulation via dual-wavelength optogenetic control. We apply this system to regulate cytoskeletal and enzymatic functions of two non-tagged endogenous proteins, actin and RAS GTPase, involved in complex functional networks sensitive to perturbations. The optogenetically-enhanced intrabodies allow fast and reversible regulation of both proteins, as well as simultaneous monitoring of RAS signaling with visible-light biosensors, enabling all-optical approach. Growing number of intrabodies should make their incorporation into optogenetic tools the versatile technology to regulate endogenous targets.


Assuntos
Optogenética , Proteínas/metabolismo , Actinas/metabolismo , Movimento Celular/efeitos da radiação , Núcleo Celular/metabolismo , Núcleo Celular/efeitos da radiação , GTP Fosfo-Hidrolases/metabolismo , Células HeLa , Humanos , Luz , Engenharia de Proteínas
12.
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
13.
Oncogene ; 39(8): 1830, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31576012

RESUMO

The original version of this Article contained an error in the author affiliations. Vladislav V. Verkhusha was incorrectly associated with the School of Mathematics, Statistics & Applied Mathematics, National University of Ireland Galway, Galway, Ireland. The correct affiliation is Anatomy and Structural Biology, Albert Einstein College of Medicine, Yeshiva University, Bronx, NY, USA.

14.
Sci Adv ; 5(12): eaay1211, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31844671

RESUMO

Focusing light deep by engineering wavefronts toward guide stars inside scattering media has potential biomedical applications in imaging, manipulation, stimulation, and therapy. However, the lack of endogenous guide stars in biological tissue hinders its translations to in vivo applications. Here, we use a reversibly switchable bacterial phytochrome protein as a genetically encoded photochromic guide star (GePGS) in living tissue to tag photons at targeted locations, achieving light focusing inside the tissue by wavefront shaping. As bacterial phytochrome-based GePGS absorbs light differently upon far-red and near-infrared illumination, a large dynamic absorption contrast can be created to tag photons inside tissue. By modulating the GePGS at a distinctive frequency, we suppressed the competition between GePGS and tissue motions and formed tight foci inside mouse tumors in vivo and acute mouse brain tissue, thus improving light delivery efficiency and specificity. Spectral multiplexing of GePGS proteins with different colors is an attractive possibility.


Assuntos
Encéfalo/diagnóstico por imagem , Imagem Molecular , Neoplasias/diagnóstico por imagem , Fitocromo/farmacologia , Animais , Pesquisa Biomédica , Encéfalo/patologia , Terapia Genética , Humanos , Luz , Camundongos , Neoplasias/patologia , Neurônios/química , Neurônios/efeitos dos fármacos , Neurônios/efeitos da radiação , Fótons , Fitocromo/química , Fitocromo/genética
15.
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.

16.
Oncogene ; 38(30): 5839-5859, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31285548

RESUMO

The cytoskeletal interacting protein Septin 9 (SEPT9), a member of the septin gene family, has been proposed to have oncogenic functions. It is a known hot spot of retroviral tagging insertion and a fusion partner of both de novo and therapy-induced mixed lineage leukemia (MLL). Of all septins, SEPT9 holds the strongest link to cancer, especially breast cancer. Murine models of breast cancer frequently exhibit SEPT9 amplification in the form of double minute chromosomes, and about 20% of human breast cancer display genomic amplification and protein over expression at the SEPT9 locus. Yet, a clear mechanism by which SEPT9 elicits tumor-promoting functions is lacking. To obtain unbiased insights on molecular signatures of SEPT9 upregulation in breast tumors, we overexpressed several of its isoforms in breast cancer cell lines. Global transcriptomic profiling supports a role of SEPT9 in invasion. Functional studies reveal that SEPT9 upregulation is sufficient to increase degradation of the extracellular matrix, while SEPT9 downregulation inhibits this process. The degradation pattern is peripheral and associated with focal adhesions (FAs), where it is coupled with increased expression of matrix metalloproteinases (MMPs). SEPT9 overexpression induces MMP upregulation in human tumors and in culture models and promotes MMP3 secretion to the media at FAs. Downregulation of SEPT9 or chemical inhibition of septin filament assembly impairs recruitment of MMP3 to FAs. Our results indicate that SEPT9 promotes upregulation and both trafficking and secretion of MMPs near FAs, thus enhancing migration and invasion of breast cancer cells.


Assuntos
Neoplasias da Mama/patologia , Carcinogênese , Movimento Celular , Matriz Extracelular/metabolismo , Adesões Focais , Glândulas Mamárias Humanas/patologia , Metaloproteinases da Matriz/metabolismo , Isoformas de Proteínas/fisiologia , Septinas/fisiologia , Neoplasias da Mama/enzimologia , Neoplasias da Mama/metabolismo , Matriz Extracelular/enzimologia , Humanos , Células MCF-7 , Glândulas Mamárias Humanas/metabolismo , Invasividade Neoplásica , Septinas/genética , Microambiente Tumoral , Regulação para Cima
17.
Nat Commun ; 10(1): 1129, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30850602

RESUMO

Optical control over the activity of receptor tyrosine kinases (RTKs) provides an efficient way to reversibly and non-invasively map their functions. We combined catalytic domains of Trk (tropomyosin receptor kinase) family of RTKs, naturally activated by neurotrophins, with photosensory core module of DrBphP bacterial phytochrome to develop opto-kinases, termed Dr-TrkA and Dr-TrkB, reversibly switchable on and off with near-infrared and far-red light. We validated Dr-Trk ability to reversibly light-control several RTK pathways, calcium level, and demonstrated that their activation triggers canonical Trk signaling. Dr-TrkA induced apoptosis in neuroblastoma and glioblastoma, but not in other cell types. Absence of spectral crosstalk between Dr-Trks and blue-light-activatable LOV-domain-based translocation system enabled intracellular targeting of Dr-TrkA independently of its activation, additionally modulating Trk signaling. Dr-Trks have several superior characteristics that make them the opto-kinases of choice for regulation of RTK signaling: high activation range, fast and reversible photoswitching, and multiplexing with visible-light-controllable optogenetic tools.


Assuntos
Fatores de Crescimento Neural/genética , Neuroglia/efeitos da radiação , Neurônios/efeitos da radiação , Fitocromo/genética , Receptor trkA/genética , Receptor trkB/genética , Animais , Apoptose/genética , Apoptose/efeitos da radiação , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular Tumoral , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Células Epiteliais/efeitos da radiação , Regulação da Expressão Gênica , Células HeLa , Humanos , Raios Infravermelhos , Transdução de Sinal Luminoso , Camundongos , Fatores de Crescimento Neural/metabolismo , Neuroglia/citologia , Neuroglia/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Optogenética/métodos , Fitocromo/metabolismo , Engenharia de Proteínas , Receptor trkA/metabolismo , Receptor trkB/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo
18.
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
19.
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
20.
Proc Natl Acad Sci U S A ; 115(50): E11681-E11690, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30478057

RESUMO

The dramatic reorganization of chromatin during mitosis is perhaps one of the most fundamental of all cell processes. It remains unclear how epigenetic histone modifications, despite their crucial roles in regulating chromatin architectures, are dynamically coordinated with chromatin reorganization in controlling this process. We have developed and characterized biosensors with high sensitivity and specificity based on fluorescence resonance energy transfer (FRET). These biosensors were incorporated into nucleosomes to visualize histone H3 Lys-9 trimethylation (H3K9me3) and histone H3 Ser-10 phosphorylation (H3S10p) simultaneously in the same live cell. We observed an anticorrelated coupling in time between H3K9me3 and H3S10p in a single live cell during mitosis. A transient increase of H3S10p during mitosis is accompanied by a decrease of H3K9me3 that recovers before the restoration of H3S10p upon mitotic exit. We further showed that H3S10p is causatively critical for the decrease of H3K9me3 and the consequent reduction of heterochromatin structure, leading to the subsequent global chromatin reorganization and nuclear envelope dissolution as a cell enters mitosis. These results suggest a tight coupling of H3S10p and H3K9me3 dynamics in the regulation of heterochromatin dissolution before a global chromatin reorganization during mitosis.


Assuntos
Técnicas Biossensoriais/métodos , Montagem e Desmontagem da Cromatina , Código das Histonas , Proteínas de Bactérias , Montagem e Desmontagem da Cromatina/genética , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas de Fluorescência Verde , Células HEK293 , Heterocromatina/genética , Heterocromatina/metabolismo , Código das Histonas/genética , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Proteínas Luminescentes , Mitose , Modelos Biológicos , Análise de Célula Única/métodos
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...