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1.
Proc Natl Acad Sci U S A ; 121(12): e2308478121, 2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38489389

RESUMO

The marine cyanobacterium Prochlorococcus is a main contributor to global photosynthesis, whilst being limited by iron availability. Cyanobacterial genomes generally encode two different types of FutA iron-binding proteins: periplasmic FutA2 ABC transporter subunits bind Fe(III), while cytosolic FutA1 binds Fe(II). Owing to their small size and their economized genome Prochlorococcus ecotypes typically possess a single futA gene. How the encoded FutA protein might bind different Fe oxidation states was previously unknown. Here, we use structural biology techniques at room temperature to probe the dynamic behavior of FutA. Neutron diffraction confirmed four negatively charged tyrosinates, that together with a neutral water molecule coordinate iron in trigonal bipyramidal geometry. Positioning of the positively charged Arg103 side chain in the second coordination shell yields an overall charge-neutral Fe(III) binding state in structures determined by neutron diffraction and serial femtosecond crystallography. Conventional rotation X-ray crystallography using a home source revealed X-ray-induced photoreduction of the iron center with observation of the Fe(II) binding state; here, an additional positioning of the Arg203 side chain in the second coordination shell maintained an overall charge neutral Fe(II) binding site. Dose series using serial synchrotron crystallography and an XFEL X-ray pump-probe approach capture the transition between Fe(III) and Fe(II) states, revealing how Arg203 operates as a switch to accommodate the different iron oxidation states. This switching ability of the Prochlorococcus FutA protein may reflect ecological adaptation by genome streamlining and loss of specialized FutA proteins.


Assuntos
Compostos Férricos , Prochlorococcus , Compostos Férricos/química , Proteínas de Ligação ao Ferro/metabolismo , Prochlorococcus/metabolismo , Ferro/metabolismo , Oxirredução , Transferrina/metabolismo , Água/química , Compostos Ferrosos/química , Cristalografia por Raios X
2.
Nat Methods ; 20(4): 541-545, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36973546

RESUMO

We report the evolution of mScarlet3, a cysteine-free monomeric red fluorescent protein with fast and complete maturation, as well as record brightness, quantum yield (75%) and fluorescence lifetime (4.0 ns). The mScarlet3 crystal structure reveals a barrel rigidified at one of its heads by a large hydrophobic patch of internal residues. mScarlet3 behaves well as a fusion tag, displays no apparent cytotoxicity and it surpasses existing red fluorescent proteins as a Förster resonance energy transfer acceptor and as a reporter in transient expression systems.


Assuntos
Transferência Ressonante de Energia de Fluorescência , Humanos , Células HeLa , Proteínas Luminescentes/metabolismo , Proteína Vermelha Fluorescente
3.
IUCrJ ; 9(Pt 6): 756-767, 2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-36381146

RESUMO

The development of serial crystallography over the last decade at XFELs and synchrotrons has produced a renaissance in room-temperature macromolecular crystallography (RT-MX), and fostered many technical and methodological breakthroughs designed to study phenomena occurring in proteins on the picosecond-to-second timescale. However, there are components of protein dynamics that occur in much slower regimes, of which the study could readily benefit from state-of-the-art RT-MX. Here, the room-temperature structural study of the relaxation of a reaction intermediate at a synchrotron, exploiting a handful of single crystals, is described. The intermediate in question is formed in microseconds during the photoreaction of the LOV2 domain of phototropin 2 from Arabidopsis thaliana, which then decays in minutes. This work monitored its relaxation in the dark using a fast-readout EIGER X 4M detector to record several complete oscillation X-ray diffraction datasets, each of 1.2 s total exposure time, at different time points in the relaxation process. Coupled with in crystallo UV-Vis absorption spectroscopy, this RT-MX approach allowed the authors to follow the relaxation of the photoadduct, a thio-ether covalent bond between the chromophore and a cysteine residue. Unexpectedly, the return of the chromophore to its spectroscopic ground state is followed by medium-scale protein rearrangements that trigger a crystal phase transition and hinder the full recovery of the structural ground state of the protein. In addition to suggesting a hitherto unexpected role of a conserved tryptophan residue in the regulation of the photocycle of LOV2, this work provides a basis for performing routine time-resolved protein crystallography experiments at synchrotrons for phenomena occurring on the second-to-hour timescale.

4.
ACS Cent Sci ; 8(1): 57-66, 2022 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-35106373

RESUMO

Optical control has enabled functional modulation in cell culture with unparalleled spatiotemporal resolution. However, current tools for in vivo manipulation are scarce. Here, we design and implement a genuine on-off optochemical probe capable of achieving hematopoietic control in zebrafish. Our photopharmacological approach first developed conformationally strained visible light photoswitches (CS-VIPs) as inhibitors of the histone methyltransferase MLL1 (KMT2A). In blood homeostasis MLL1 plays a crucial yet controversial role. CS-VIP 8 optimally fulfils the requirements of a true bistable functional system in vivo under visible-light irradiation, and with unprecedented stability. These properties are exemplified via hematopoiesis photoinhibition with a single isomer in zebrafish. The present interdisciplinary study uncovers the mechanism of action of CS-VIPs. Upon WDR5 binding, CS-VIP 8 causes MLL1 release with concomitant allosteric rearrangements in the WDR5/RbBP5 interface. Since our tool provides on-demand reversible control without genetic intervention or continuous irradiation, it will foster hematopathology and epigenetic investigations. Furthermore, our workflow will enable exquisite photocontrol over other targets inhibited by macrocycles.

5.
Nat Commun ; 12(1): 4542, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34315871

RESUMO

Folate enzyme cofactors and their derivatives have the unique ability to provide a single carbon unit at different oxidation levels for the de novo synthesis of amino-acids, purines, or thymidylate, an essential DNA nucleotide. How these cofactors mediate methylene transfer is not fully settled yet, particularly with regard to how the methylene is transferred to the methylene acceptor. Here, we uncovered that the bacterial thymidylate synthase ThyX, which relies on both folate and flavin for activity, can also use a formaldehyde-shunt to directly synthesize thymidylate. Combining biochemical, spectroscopic and anaerobic crystallographic analyses, we showed that formaldehyde reacts with the reduced flavin coenzyme to form a carbinolamine intermediate used by ThyX for dUMP methylation. The crystallographic structure of this intermediate reveals how ThyX activates formaldehyde and uses it, with the assistance of active site residues, to methylate dUMP. Our results reveal that carbinolamine species promote methylene transfer and suggest that the use of a CH2O-shunt may be relevant in several other important folate-dependent reactions.


Assuntos
Formaldeído/metabolismo , Nucleotídeos/metabolismo , Thermotoga maritima/enzimologia , Timidilato Sintase/metabolismo , Biocatálise , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Domínio Catalítico , Ativação Enzimática , Flavinas/metabolismo , Metilação , Eletricidade Estática , Timidilato Sintase/química
6.
PLoS Biol ; 18(11): e3000936, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33137097

RESUMO

Using mRNA sequencing and de novo transcriptome assembly, we identified, cloned, and characterized 9 previously undiscovered fluorescent protein (FP) homologs from Aequorea victoria and a related Aequorea species, with most sequences highly divergent from A. victoria green fluorescent protein (avGFP). Among these FPs are the brightest green fluorescent protein (GFP) homolog yet characterized and a reversibly photochromic FP that responds to UV and blue light. Beyond green emitters, Aequorea species express purple- and blue-pigmented chromoproteins (CPs) with absorbances ranging from green to far-red, including 2 that are photoconvertible. X-ray crystallography revealed that Aequorea CPs contain a chemically novel chromophore with an unexpected crosslink to the main polypeptide chain. Because of the unique attributes of several of these newly discovered FPs, we expect that Aequorea will, once again, give rise to an entirely new generation of useful probes for bioimaging and biosensing.


Assuntos
Hidrozoários/genética , Hidrozoários/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Animais , Técnicas Biossensoriais , Cor , Cristalografia por Raios X , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hidrozoários/química , Proteínas Luminescentes/química , Modelos Moleculares , Imagem Óptica , Filogenia , Eletricidade Estática
7.
Nat Commun ; 11(1): 2137, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358514

RESUMO

The light-driven sodium-pumping rhodopsin KR2 from Krokinobacter eikastus is the only non-proton cation active transporter with demonstrated potential for optogenetics. However, the existing structural data on KR2 correspond exclusively to its ground state, and show no sodium inside the protein, which hampers the understanding of sodium-pumping mechanism. Here we present crystal structure of the O-intermediate of the physiologically relevant pentameric form of KR2 at the resolution of 2.1 Å, revealing a sodium ion near the retinal Schiff base, coordinated by N112 and D116 of the characteristic NDQ triad. We also obtained crystal structures of D116N and H30A variants, conducted metadynamics simulations and measured pumping activities of putative pathway mutants to demonstrate that sodium release likely proceeds alongside Q78 towards the structural sodium ion bound between KR2 protomers. Our findings highlight the importance of pentameric assembly for sodium pump function, and may be used for rational engineering of enhanced optogenetic tools.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Flavobacteriaceae/metabolismo , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/metabolismo , Cristalografia por Raios X , Escherichia coli/metabolismo , Simulação de Dinâmica Molecular , Dobramento de Proteína , Rodopsina/química , Rodopsina/metabolismo , Sódio/metabolismo , Difração de Raios X
8.
IUCrJ ; 6(Pt 4): 665-680, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31316810

RESUMO

Carrying out macromolecular crystallography (MX) experiments at cryogenic temperatures significantly slows the rate of global radiation damage, thus facilitating the solution of high-resolution crystal structures of macromolecules. However, cryo-MX experiments suffer from the early onset of so-called specific radiation damage that affects certain amino-acid residues and, in particular, the active sites of many proteins. Here, a series of MX experiments are described which suggest that specific and global radiation damage are much less decoupled at room temperature than they are at cryogenic temperatures. The results reported here demonstrate the interest in reviving the practice of collecting MX diffraction data at room temperature and allow structural biologists to favourably envisage the development of time-resolved MX experiments at synchrotron sources.

9.
Nat Methods ; 14(1): 53-56, 2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27869816

RESUMO

We report the engineering of mScarlet, a truly monomeric red fluorescent protein with record brightness, quantum yield (70%) and fluorescence lifetime (3.9 ns). We developed mScarlet starting with a consensus synthetic template and using improved spectroscopic screening techniques; mScarlet's crystal structure reveals a planar and rigidified chromophore. mScarlet outperforms existing red fluorescent proteins as a fusion tag, and it is especially useful as a Förster resonance energy transfer (FRET) acceptor in ratiometric imaging.


Assuntos
Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Proteínas Luminescentes/metabolismo , Imagem Molecular/métodos , Engenharia de Proteínas/métodos , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Sobrevivência Celular , Células HeLa , Humanos , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Células Tumorais Cultivadas , Proteína Vermelha Fluorescente
10.
Protein Sci ; 25(2): 308-15, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26549191

RESUMO

Fluorescent proteins (FPs) are powerful tools for cell and molecular biology. Here based on structural analysis, a blue-shifted mutant of a recently engineered monomeric infrared fluorescent protein (mIFP) has been rationally designed. This variant, named iBlueberry, bears a single mutation that shifts both excitation and emission spectra by approximately 40 nm. Furthermore, iBlueberry is four times more photostable than mIFP, rendering it more advantageous for imaging protein dynamics. By tagging iBlueberry to centrin, it has been demonstrated that the fusion protein labels the centrosome in the developing zebrafish embryo. Together with GFP-labeled nucleus and tdTomato-labeled plasma membrane, time-lapse imaging to visualize the dynamics of centrosomes in radial glia neural progenitors in the intact zebrafish brain has been demonstrated. It is further shown that iBlueberry can be used together with mIFP in two-color protein labeling in living cells and in two-color tumor labeling in mice.


Assuntos
Corantes Fluorescentes/análise , Proteínas Luminescentes/análise , Imagem Óptica/métodos , Animais , Linhagem Celular Tumoral , Drosophila/ultraestrutura , Feminino , Corantes Fluorescentes/metabolismo , Células HEK293 , Humanos , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação , Neoplasias/diagnóstico , Fotodegradação , Conformação Proteica , Estabilidade Proteica , Peixe-Zebra/embriologia , Proteína Vermelha Fluorescente
11.
Nat Commun ; 5: 3626, 2014 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-24832154

RESUMO

Infrared fluorescent proteins (IFPs) are ideal for in vivo imaging, and monomeric versions of these proteins can be advantageous as protein tags or for sensor development. In contrast to GFP, which requires only molecular oxygen for chromophore maturation, phytochrome-derived IFPs incorporate biliverdin (BV) as the chromophore. However, BV varies in concentration in different cells and organisms. Here we engineered cells to express the haeme oxygenase responsible for BV biosynthesis and a brighter monomeric IFP mutant (IFP2.0). Together, these tools improve the imaging capabilities of IFP2.0 compared with monomeric IFP1.4 and dimeric iRFP. By targeting IFP2.0 to the plasma membrane, we demonstrate robust labelling of neuronal processes in Drosophila larvae. We also show that this strategy improves the sensitivity when imaging brain tumours in whole mice. Our work shows promise in the application of IFPs for protein labelling and in vivo imaging.


Assuntos
Neoplasias Encefálicas/diagnóstico , Corantes Fluorescentes/metabolismo , Proteínas Luminescentes/metabolismo , Neuroimagem/métodos , Neurônios/metabolismo , Animais , Biliverdina/metabolismo , Neoplasias Encefálicas/metabolismo , Cristalografia por Raios X , Drosophila , Células HEK293 , Heme Oxigenase (Desciclizante)/metabolismo , Humanos , Raios Infravermelhos , Larva , Camundongos , Microscopia Confocal , Fitocromo , Ratos
12.
J Struct Biol ; 181(2): 89-94, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23178456

RESUMO

Selenomethionine labeling is the most common technique used in protein crystallography to derivatize recombinant proteins for experimental phasing using anomalous scattering at tunable synchrotron beamlines. Recently, it has been shown that UV radiation depletes electron density of selenium atoms of selenomethionine residues and that UV radiation-damage-induced phasing (equivalent to single isomorphous replacement) protocol can be applied to calculate experimental phases. Here we present the straightforward integration of a UV source with an in-house diffractometer. We show how this setup can extend the capabilities of a sealed tube X-ray generator and be used for experimental phasing of selenium-labeled proteins.


Assuntos
Cristalografia/métodos , Modelos Moleculares , Proteínas Recombinantes/análise , Selenometionina/química , Raios Ultravioleta , Lasers , Proteínas Recombinantes/química , Difração de Raios X/métodos
13.
Nat Commun ; 3: 751, 2012 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-22434194

RESUMO

Cyan variants of green fluorescent protein are widely used as donors in Förster resonance energy transfer experiments. The popular, but modestly bright, Enhanced Cyan Fluorescent Protein (ECFP) was sequentially improved into the brighter variants Super Cyan Fluorescent Protein 3A (SCFP3A) and mTurquoise, the latter exhibiting a high-fluorescence quantum yield and a long mono-exponential fluorescence lifetime. Here we combine X-ray crystallography and excited-state calculations to rationalize these stepwise improvements. The enhancement originates from stabilization of the seventh ß-strand and the strengthening of the sole chromophore-stabilizing hydrogen bond. The structural analysis highlighted one suboptimal internal residue, which was subjected to saturation mutagenesis combined with fluorescence lifetime-based screening. This resulted in mTurquoise2, a brighter variant with faster maturation, high photostability, longer mono-exponential lifetime and the highest quantum yield measured for a monomeric fluorescent protein. Together, these properties make mTurquoise2 the preferable cyan variant of green fluorescent protein for long-term imaging and as donor for Förster resonance energy transfer to a yellow fluorescent protein.


Assuntos
Proteínas de Fluorescência Verde/química , Proteínas de Bactérias/química , Linhagem Celular Tumoral , Cristalografia por Raios X/métodos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/ultraestrutura , Células HeLa , Humanos , Ligação de Hidrogênio , Mutagênese Sítio-Dirigida , Conformação Proteica , Estrutura Secundária de Proteína
14.
Science ; 324(5928): 804-7, 2009 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-19423828

RESUMO

Visibly fluorescent proteins (FPs) from jellyfish and corals have revolutionized many areas of molecular and cell biology, but the use of FPs in intact animals, such as mice, has been handicapped by poor penetration of excitation light. We now show that a bacteriophytochrome from Deinococcus radiodurans, incorporating biliverdin as the chromophore, can be engineered into monomeric, infrared-fluorescent proteins (IFPs), with excitation and emission maxima of 684 and 708 nm, respectively; extinction coefficient >90,000 M(-1) cm(-1); and quantum yield of 0.07. IFPs express well in mammalian cells and mice and spontaneously incorporate biliverdin, which is ubiquitous as the initial intermediate in heme catabolism but has negligible fluorescence by itself. Because their wavelengths penetrate tissue well, IFPs are suitable for whole-body imaging. The IFPs developed here provide a scaffold for further engineering.


Assuntos
Biliverdina , Deinococcus/química , Proteínas Luminescentes , Fitocromo , Engenharia de Proteínas , Adenoviridae/genética , Sequência de Aminoácidos , Animais , Biliverdina/química , Biliverdina/metabolismo , Linhagem Celular , Diagnóstico por Imagem , Fluorescência , Humanos , Fígado/anatomia & histologia , Proteínas Luminescentes/química , Proteínas Luminescentes/metabolismo , Camundongos , Dados de Sequência Molecular , Fitocromo/química , Fitocromo/genética , Fitocromo/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Espectrofotometria Infravermelho , Imagem Corporal Total
15.
J Synchrotron Radiat ; 14(Pt 1): 99-108, 2007 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-17211076

RESUMO

High-quality Raman spectra have been collected on single DNA crystals kept at 100 K in a flow of cold nitrogen gas prior, during and after macromolecular crystallography (MX) data collection. The non-symmetric bending vibration for C-Br bonds in 8-bromo-2'-deoxyguanosine has been unambiguously identified at 293 cm(-1) in the non-resonant Raman spectra. The breakage of this bond could be monitored by the continuous recording of Raman spectra during X-ray exposure, and its decay versus dose could be directly correlated with the loss of the bromide definition within the electron-density maps as determined with MX.


Assuntos
Cristalografia/métodos , Dano ao DNA , DNA/química , DNA/efeitos da radiação , Modelos Químicos , Modelos Moleculares , Análise Espectral Raman/métodos , Simulação por Computador , DNA/ultraestrutura , Relação Dose-Resposta à Radiação , Desnaturação de Ácido Nucleico/efeitos da radiação , Sistemas On-Line , Doses de Radiação , Raios X
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