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1.
Int J Mol Sci ; 22(19)2021 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-34639031

RESUMO

The family of phytochrome photoreceptors contains proteins with different domain architectures and spectral properties. Knotless phytochromes are one of the three main subgroups classified by their distinct lack of the PAS domain in their photosensory core module, which is in contrast to the canonical PAS-GAF-PHY array. Despite intensive research on the ultrafast photodynamics of phytochromes, little is known about the primary kinetics in knotless phytochromes. Here, we present the ultrafast Pr ⇆ Pfr photodynamics of SynCph2, the best-known knotless phytochrome. Our results show that the excited state lifetime of Pr* (~200 ps) is similar to bacteriophytochromes, but much longer than in most canonical phytochromes. We assign the slow Pr* kinetics to relaxation processes of the chromophore-binding pocket that controls the bilin chromophore's isomerization step. The Pfr photoconversion dynamics starts with a faster excited state relaxation than in canonical phytochromes, but, despite the differences in the respective domain architectures, proceeds via similar ground state intermediate steps up to Meta-F. Based on our observations, we propose that the kinetic features and overall dynamics of the ultrafast photoreaction are determined to a great extent by the geometrical context (i.e., available space and flexibility) within the binding pocket, while the general reaction steps following the photoexcitation are most likely conserved among the red/far-red phytochromes.


Assuntos
Processos Fotoquímicos , Fitocromo/química , Fitocromo/metabolismo , Cinética , Luz , Modelos Moleculares , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/metabolismo , Conformação Proteica , Análise Espectral , Relação Estrutura-Atividade
2.
Phys Chem Chem Phys ; 23(37): 20867-20874, 2021 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-34374395

RESUMO

Cyanobacteriochromes (CBCRs) are bi-stable photoreceptor proteins with high potential for biotechnological applications. Most of these proteins utilize phycocyanobilin (PCB) as a light-sensing co-factor, which is unique to cyanobacteria, but some variants also incorporate biliverdin (BV). The latter are of particular interest for biotechnology due to the natural abundance and red-shifted absorption of BV. Here, AmI-g2 was investigated, a CBCR capable of binding both PCB and BV. The assembly kinetics and primary photochemistry of AmI-g2 with both chromophores were studied in vitro. The assembly reaction with PCB is roughly 10× faster than BV, and the formation of a non-covalent intermediate was identified as the rate-limiting step in the case of BV. This step is fast for PCB, where the formation of the covalent thioether bond between AmI-g2 and PCB becomes rate-limiting. The photochemical quantum yields of the forward and backward reactions of AmI-g2 were estimated and discussed in the context of homologous CBCRs.


Assuntos
Biliverdina/química , Cianobactérias/metabolismo , Fotorreceptores Microbianos/química , Ficobilinas/química , Ficocianina/química , Biliverdina/metabolismo , Cinética , Fotorreceptores Microbianos/genética , Fotorreceptores Microbianos/metabolismo , Ficobilinas/metabolismo , Ficocianina/metabolismo , Ligação Proteica , Teoria Quântica , Espectrofotometria
3.
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
4.
Nat Commun ; 12(1): 4394, 2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34285211

RESUMO

Bacterial phytochrome photoreceptors usually belong to two-component signaling systems which transmit environmental stimuli to a response regulator through a histidine kinase domain. Phytochromes switch between red light-absorbing and far-red light-absorbing states. Despite exhibiting extensive structural responses during this transition, the model bacteriophytochrome from Deinococcus radiodurans (DrBphP) lacks detectable kinase activity. Here, we resolve this long-standing conundrum by comparatively analyzing the interactions and output activities of DrBphP and a bacteriophytochrome from Agrobacterium fabrum (Agp1). Whereas Agp1 acts as a conventional histidine kinase, we identify DrBphP as a light-sensitive phosphatase. While Agp1 binds its cognate response regulator only transiently, DrBphP does so strongly, which is rationalized at the structural level. Our data pinpoint two key residues affecting the balance between kinase and phosphatase activities, which immediately bears on photoreception and two-component signaling. The opposing output activities in two highly similar bacteriophytochromes suggest the use of light-controllable histidine kinases and phosphatases for optogenetics.


Assuntos
Proteínas de Bactérias/metabolismo , Histidina Quinase/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fotorreceptores Microbianos/metabolismo , Transdução de Sinais/efeitos da radiação , Agrobacterium/enzimologia , Proteínas de Bactérias/ultraestrutura , Deinococcus/enzimologia , Histidina Quinase/ultraestrutura , Luz , Simulação de Dinâmica Molecular , Monoéster Fosfórico Hidrolases/ultraestrutura , Fotorreceptores Microbianos/ultraestrutura , Domínios Proteicos
5.
Angew Chem Int Ed Engl ; 60(34): 18688-18693, 2021 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-34097335

RESUMO

Phytochrome proteins are light receptors that play a pivotal role in regulating the life cycles of plants and microorganisms. Intriguingly, while cyanobacterial phytochrome Cph1 and cyanobacteriochrome AnPixJ use the same phycocyanobilin (PCB) chromophore to absorb light, their excited-state behavior is very different. We employ multiscale calculations to rationalize the different early photoisomerization mechanisms of PCB in Cph1 and AnPixJ. We found that their electronic S1 , T1 , and S0 potential minima exhibit distinct geometric and electronic structures due to different hydrogen bond networks with the protein environment. These specific interactions influence the S1 electronic structures along the photoisomerization paths, ultimately leading to internal conversion in Cph1 but intersystem crossing in AnPixJ. This explains why the excited-state relaxation in AnPixJ is much slower (ca. 100 ns) than in Cph1 (ca. 30 ps). Further, we predict that efficient internal conversion in AnPixJ can be achieved upon protonating the carboxylic group that interacts with PCB.


Assuntos
Proteínas de Bactérias/química , Cianobactérias/química , Fotorreceptores Microbianos/química , Ficobilinas/química , Ficocianina/química , Fitocromo/química , Proteínas Quinases/química , Proteínas de Bactérias/metabolismo , Cianobactérias/metabolismo , Ligação de Hidrogênio , Estrutura Molecular , Processos Fotoquímicos , Fotorreceptores Microbianos/metabolismo , Ficobilinas/metabolismo , Ficocianina/metabolismo , Fitocromo/metabolismo , Proteínas Quinases/metabolismo , Estereoisomerismo
6.
Photochem Photobiol Sci ; 20(3): 451-473, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33721277

RESUMO

Bacteria and fungi of the plant microbiota can be phytopathogens, parasites or symbionts that establish mutually advantageous relationships with plants. They are often rich in photoreceptors for UVA-Visible light, and in many cases, they exhibit light regulation of growth patterns, infectivity or virulence, reproductive traits, and production of pigments and of metabolites. In addition to the light-driven effects, often demonstrated via the generation of photoreceptor gene knock-outs, microbial photoreceptors can exert effects also in the dark. Interestingly, some fungi switch their attitude towards plants in dependence of illumination or dark conditions in as much as they may be symbiotic or pathogenic. This review summarizes the current knowledge about the roles of light and photoreceptors in plant-associated bacteria and fungi aiming at the identification of common traits and general working ideas. Still, reports on light-driven infection of plants are often restricted to the description of macroscopically observable phenomena, whereas detailed information on the molecular level, e.g., protein-protein interaction during signal transduction or induction mechanisms of infectivity/virulence initiation remains sparse. As it becomes apparent from still only few molecular studies, photoreceptors, often from the red- and the blue light sensitive groups interact and mutually modulate their individual effects. The topic is of great relevance, even in economic terms, referring to plant-pathogen or plant-symbionts interactions, considering the increasing usage of artificial illumination in greenhouses, the possible light-regulation of the synthesis of plant-growth stimulating substances or herbicides by certain symbionts, and the biocontrol of pests by selected fungi and bacteria in a sustainable agriculture.


Assuntos
Luz , Microbiota/efeitos da radiação , Plantas/microbiologia , Bactérias/metabolismo , Bactérias/patogenicidade , Fungos/metabolismo , Fungos/patogenicidade , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/metabolismo , Fitocromo/química , Fitocromo/metabolismo
7.
Biochim Biophys Acta Proteins Proteom ; 1869(5): 140620, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33561578

RESUMO

The aggregation of proteins is of importance in fields ranging from protein homeostasis to disease. The light-sensing protein Vivid (VVD) regulates responses to blue-light illumination in the filamentous fungus Neurospora crassa. Consisting of a single light­oxygen-voltage domain, VVD is characterized by cycling between dark and lit states that correspond to formation and disruption of a photoadduct between the flavin cofactor and the apoprotein. Recently, in vitro assays have shown that VVD undergoes self-oxidative damage and aggregation resulting from excessive blue-light illumination. To explore the aggregation process of VVD, here we study the kinetics of aggregation and how it is influenced by environmental factors such as initial protein concentration, temperature, and light. We found that the aggregation kinetics of VVD is consistent with a second-order reaction model involving kinetic control, where thermal decay from lit-VVD to dark-VVD is necessary for aggregation to proceed. The height of the energy barrier separating the lit and dark VVD states is measured as (80 ± 2) kJ mol-1. Application of the kinetic model to the observed dependence of aggregation vs. temperature allowed us to further estimate the energy involved in the nucleation of dark-VVD, (257 ± 24) kJ mol-1. Finally, we show that VVD aggregation levels increase as the time of blue light exposure is augmented, suggesting possible mechanisms for protein damage. These results demonstrate how aggregation of a photoreceptor depends not only on environmental factors but on the intrinsic response of the protein to illumination.


Assuntos
Proteínas Fúngicas/metabolismo , Neurospora crassa/metabolismo , Fotorreceptores Microbianos/metabolismo , Escuridão , Dimerização , Cinética , Luz , Modelos Químicos
8.
Biochem J ; 478(5): 1043-1059, 2021 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-33559683

RESUMO

Cyanobacteriochromes are linear tetrapyrrole-binding photoreceptors produced by cyanobacteria. Their chromophore-binding GAF domains are categorized into many lineages. Among them, dual Cys-type cyanobacteriochrome GAF domains possessing not only a highly conserved 'first Cys' but also a 'second Cys' are found from multiple lineages. The first Cys stably attaches to C31 of the A-ring, while the second Cys mostly shows reversible ligation to the C10 of the chromophore. Notably, the position of the second Cys in the primary sequence is diversified, and the most abundant dual Cys-type GAF domains have a 'second Cys' within the DXCF motif, which are called DXCF GAF domains. It has been long known that the second Cys in the DXCF GAF domains not only shows the reversible ligation but also is involved in isomerization activity (reduction in C4=C5 double bond) from the initially incorporated phycocyanobilin to phycoviolobilin. However, comprehensive site-directed mutagenesis on the DXCF GAF domains, AM1_6305g1 and AM1_1499g1, revealed that the second Cys is dispensable for isomerization activity, in which three residues participate by fixing the C- and D-rings. Fixation of the chromophore on both sides of the C5 bridge is necessary, even though one side of the fixation site is far from this bridge, with the other side at C31 fixed by the first Cys.


Assuntos
Cianobactérias/metabolismo , Cisteína/química , Mutação , Fotorreceptores Microbianos/metabolismo , Ficobilinas/biossíntese , Fitocromo/metabolismo , Cisteína/genética , Cisteína/metabolismo , Mutagênese Sítio-Dirigida , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/genética , Fitocromo/química , Fitocromo/genética , Conformação Proteica , Domínios Proteicos
9.
J Gen Appl Microbiol ; 67(2): 54-58, 2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-33342920

RESUMO

Phototaxis is a phenomenon where cyanobacteria move toward a light source. Previous studies have shown that the blue-light-using-flavin (BLUF)-type photoreceptor PixD and the response regulator-like protein PixE control the phototaxis in the cyanobacterium Synechocystis sp. PCC 6803. The pixD-null mutant moves away from light, whereas WT, pixE mutant, and pixD pixE double mutant move toward the light. This indicates that PixE functions downstream of PixD and influences the direction of movement. However, it is still unclear how the light signal received by PixD is transmitted to PixE, and then subsequently transmitted to the type IV pili motor mechanism. Here, we investigated intracellular localization and oligomerization of PixD and PixE to elucidate mechanisms of phototaxis regulation. Blue-native PAGE analysis, coupled with western blotting, indicated that most PixD exist as a dimer in soluble fractions, whereas PixE localized in ~250 kDa and ~450 kDa protein complexes in membrane fractions. When blue-native PAGE was performed after illuminating the membrane fractions with blue light, PixE levels in the ~250 kDa and ~450 kDa complexes were reduced and increased, respectively. These results suggest that PixE, localized in the ~450 kDa complex, controls activity of the motor ATPase PilB1 to regulate pilus motility.


Assuntos
Proteínas de Bactérias/metabolismo , Fotorreceptores Microbianos/metabolismo , Synechocystis/fisiologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Membrana Celular/metabolismo , Luz , Transdução de Sinal Luminoso , Modelos Biológicos , Mutação , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/genética , Fototaxia , Multimerização Proteica
10.
Biochemistry ; 59(50): 4703-4710, 2020 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-33287544

RESUMO

YtvA from Bacillus subtilis is a sensor protein that responds to blue light stress and regulates the activity of transcription factor σB. It is composed of the N-terminal LOV (light-oxygen-voltage) domain, the C-terminal STAS (sulfate transporter and anti-sigma factor antagonist) domain, and a linker region connecting them. In this study, the photoreaction and kinetics of full-length YtvA and the intermolecular interaction with a downstream protein, RsbRA, were revealed by the transient grating method. Although N-YLOV-linker, which is composed of the LOV domain of YtvA with helices A'α and Jα, exhibits a diffusion change due to the rotational motion of the helices, the YtvA dimer does not show the diffusion change. This result suggests that the STAS domain inhibits the rotational movement of helices A'α and Jα. We found that the YtvA dimer formed a heterotetramer with the RsbRA dimer probably via the interaction between the STAS domains, and we showed the diffusion change upon blue light illumination with a time constant faster than 70 µs. This result suggests a conformational change of the STAS domains; i.e., the interface between the STAS domains of the proteins changes to enhance the friction with water by the rotation structural change of helices A'α and Jα of YtvA.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/efeitos da radiação , Fosfoproteínas/química , Fosfoproteínas/efeitos da radiação , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/efeitos da radiação , Bacillus subtilis/química , Bacillus subtilis/metabolismo , Bacillus subtilis/efeitos da radiação , Proteínas de Bactérias/metabolismo , Difusão Dinâmica da Luz , Luz , Modelos Moleculares , Fosfoproteínas/metabolismo , Processos Fotoquímicos , Fotorreceptores Microbianos/metabolismo , Domínios e Motivos de Interação entre Proteínas/efeitos da radiação , Estrutura Quaternária de Proteína/efeitos da radiação
11.
Biochemistry ; 59(41): 4015-4028, 2020 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-33021375

RESUMO

In this report, we compare the femtosecond to nanosecond primary reverse photodynamics (15EPg → 15ZPr) of eight tetrapyrrole binding photoswitching cyanobacteriochromes in the canonical red/green family from the cyanobacterium Nostoc punctiforme. Three characteristic classes were identified on the basis of the diversity of excited-state and ground-state properties, including the lifetime, photocycle initiation quantum yield, photointermediate stability, spectra, and temporal properties. We observed a correlation between the excited-state lifetime and peak wavelength of the electronic absorption spectrum with higher-energy-absorbing representatives exhibiting both faster excited-state decay times and higher photoisomerization quantum yields. The latter was attributed to both an increased number of structural restraints and differences in H-bonding networks that facilitate photoisomerization. All three classes exhibited primary Lumi-Go intermediates, with class II and III representatives evolving to a secondary Meta-G photointermediate. Class II Meta-GR intermediates were orange absorbing, whereas class III Meta-G had structurally relaxed, red-absorbing chromophores that resemble their dark-adapted 15ZPr states. Differences in the reverse and forward reaction mechanisms are discussed within the context of structural constraints.


Assuntos
Proteínas de Bactérias/metabolismo , Nostoc/metabolismo , Cinética , Processos Fotoquímicos , Fotorreceptores Microbianos/metabolismo
12.
Mol Plant Pathol ; 21(12): 1606-1619, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33029921

RESUMO

Adaptation and efficient colonization of the phyllosphere are essential processes for the switch to an epiphytic stage in foliar bacterial pathogens. Here, we explore the interplay among light perception and global transcriptomic alterations in epiphytic populations of the hemibiotrophic pathogen Pseudomonas syringae pv. tomato DC3000 (PsPto) following contact with tomato leaves. We found that blue-light perception by PsPto on leaf surfaces is required for optimal colonization. Blue light triggers the activation of metabolic activity and increases the transcript levels of five chemoreceptors through the function of light oxygen voltage and BphP1 photoreceptors. The inactivation of PSPTO_1008 and PSPTO_2526 chemoreceptors causes a reduction in virulence. Our results indicate that during PsPto interaction with tomato plants, light perception, chemotaxis, and virulence are highly interwoven processes.


Assuntos
Proteínas de Bactérias/metabolismo , Lycopersicon esculentum/microbiologia , Fotorreceptores Microbianos/metabolismo , Doenças das Plantas/microbiologia , Pseudomonas syringae/efeitos da radiação , Transcriptoma/efeitos da radiação , Proteínas de Bactérias/genética , Quimiotaxia/efeitos da radiação , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Luz , Fotorreceptores Microbianos/genética , Folhas de Planta/microbiologia , Folhas de Planta/efeitos da radiação , Pseudomonas syringae/genética , Pseudomonas syringae/patogenicidade , Pseudomonas syringae/fisiologia , Virulência/efeitos da radiação
13.
Proc Natl Acad Sci U S A ; 117(43): 26626-26632, 2020 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-33037153

RESUMO

Blue light using flavin (BLUF) photoreceptor proteins are critical for many light-activated biological processes and are promising candidates for optogenetics because of their modular nature and long-range signaling capabilities. Although the photocycle of the Slr1694 BLUF domain has been characterized experimentally, the identity of the light-adapted state following photoexcitation of the bound flavin remains elusive. Herein hybrid quantum mechanical/molecular mechanical (QM/MM) molecular dynamics simulations of this photocycle provide a nonequilibrium dynamical picture of a possible mechanism for the formation of the light-adapted state. Photoexcitation of the flavin induces a forward proton-coupled electron transfer (PCET) process that leads to the formation of an imidic acid tautomer of Gln50. The calculations herein show that the subsequent rotation of Gln50 allows a reverse PCET process that retains this tautomeric form. In the resulting purported light-adapted state, the glutamine tautomer forms a hydrogen bond with the flavin carbonyl group. Additional ensemble-averaged QM/MM calculations of the dark-adapted and purported light-adapted states demonstrate that the light-adapted state with the imidic acid glutamine tautomer reproduces the experimentally observed spectroscopic signatures. Specifically, the calculations reproduce the red shifts in the flavin electronic absorption and carbonyl stretch infrared spectra in the light-adapted state. Further hydrogen-bonding analyses suggest the formation of hydrogen-bonding interactions between the flavin and Arg65 in the light-adapted state, providing a plausible explanation for the experimental observation of faster photoinduced PCET in this state. These characteristics of the light-adapted state may also be essential for the long-range signaling capabilities of this photoreceptor protein.


Assuntos
Flavinas , Glutamina , Teoria da Densidade Funcional , Flavinas/química , Flavinas/metabolismo , Flavoproteínas/química , Flavoproteínas/metabolismo , Glutamina/química , Glutamina/metabolismo , Ligação de Hidrogênio , Isomerismo , Luz , Simulação de Dinâmica Molecular , Processos Fotoquímicos , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/metabolismo
14.
J Bacteriol ; 202(20)2020 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-32967908

RESUMO

Pseudomonas putida KT2440 retains three homologs (PplR1 to PplR3) of the LitR/CarH family, an adenosyl B12-dependent light-sensitive MerR family transcriptional regulator. Transcriptome analysis revealed the existence of a number of photoinducible genes, including pplR1, phrB (encoding DNA photolyase), ufaM (furan-containing fatty acid synthase), folE (GTP cyclohydrolase I), cryB (cryptochrome-like protein), and multiple genes without annotated/known function. Transcriptional analysis by quantitative reverse transcription-PCR with knockout mutants of pplR1 to pplR3 showed that a triple knockout completely abolished the light-inducible transcription in P. putida, which indicates the occurrence of ternary regulation of PplR proteins. A DNase I footprint assay showed that PplR1 protein specifically binds to the promoter regions of light-inducible genes, suggesting a consensus PplR1-binding direct repeat, 5'-T(G/A)TACAN12TGTA(C/T)A-3'. The disruption of B12 biosynthesis cluster did not affect the light-inducible transcription; however, disruption of ppSB1-LOV (where LOV indicates "light, oxygen, or voltage") and ppSB2-LOV, encoding blue light photoreceptors adjacently located to pplR3 and pplR2, respectively, led to the complete loss of light-inducible transcription. Overall, the results suggest that the three PplRs and two PpSB-LOVs cooperatively regulate the light-inducible gene expression. The wide distribution of the pplR/ppSB-LOV cognate pair homologs in Pseudomonas spp. and related bacteria suggests that the response and adaptation to light are similarly regulated in the group of nonphototrophic bacteria.IMPORTANCE The LitR/CarH family is a new group of photosensor homologous to MerR-type transcriptional regulators. Proteins of this family are distributed to various nonphototrophic bacteria and grouped into at least five classes (I to V). Pseudomonas putida retaining three class II LitR proteins exhibited a genome-wide response to light. All three paralogs were functional and mediated photodependent activation of promoters directing the transcription of light-induced genes or operons. Two LOV (light, oxygen, or voltage) domain proteins, adjacently encoded by two litR genes, were also essential for the photodependent transcriptional control. Despite the difference in light-sensing mechanisms, the DNA binding consensus of class II LitR [T(G/A)TA(C/T)A] was the same as that of class I. This is the first study showing the actual involvement of class II LitR in light-induced transcription.


Assuntos
Proteínas de Bactérias/metabolismo , Luz , Fotorreceptores Microbianos/metabolismo , Pseudomonas putida/metabolismo , Pseudomonas putida/efeitos da radiação , Proteínas de Bactérias/genética , Sítios de Ligação , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Óperon , Fotorreceptores Microbianos/genética , Regiões Promotoras Genéticas , Pseudomonas putida/genética
15.
Int J Mol Sci ; 21(17)2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32872628

RESUMO

Cyanobacteriochromes (CBCRs), which are known as linear tetrapyrrole-binding photoreceptors, to date can only be detected from cyanobacteria. They can perceive light only in a small unit, which is categorized into various lineages in correlation with their spectral and structural characteristics. Recently, we have succeeded in identifying specific molecules, which can incorporate mammalian intrinsic biliverdin (BV), from the expanded red/green (XRG) CBCR lineage and in converting BV-rejective molecules into BV-acceptable ones with the elucidation of the structural basis. Among the BV-acceptable molecules, AM1_1870g3_BV4 shows a spectral red-shift in comparison with other molecules, while NpF2164g5_BV4 does not show photoconversion but stably shows a near-infrared (NIR) fluorescence. In this study, we found that AM1_1870g3_BV4 had a specific Tyr residue near the d-ring of the chromophore, while others had a highly conserved Leu residue. The replacement of this Tyr residue with Leu in AM1_1870g3_BV4 resulted in a blue-shift of absorption peak. In contrast, reverse replacement in NpF2164g5_BV4 resulted in a red-shift of absorption and fluorescence peaks, which applies to fluorescence bio-imaging in mammalian cells. Notably, the same Tyr/Leu-dependent color-tuning is also observed for the CBCRs belonging to the other lineage, which indicates common molecular mechanisms.


Assuntos
Proteínas de Bactérias/metabolismo , Biliverdina/metabolismo , Cianobactérias/metabolismo , Fotorreceptores Microbianos/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Biliverdina/química , Cor , Células HeLa , Humanos , Luz , Homologia de Sequência
16.
Nat Commun ; 11(1): 4248, 2020 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-32843623

RESUMO

Femtosecond time-resolved crystallography (TRC) on proteins enables resolving the spatial structure of short-lived photocycle intermediates. An open question is whether confinement and lower hydration of the proteins in the crystalline state affect the light-induced structural transformations. Here, we measured the full photocycle dynamics of a signal transduction protein often used as model system in TRC, Photoactive Yellow Protein (PYP), in the crystalline state and compared those to the dynamics in solution, utilizing electronic and vibrational transient absorption measurements from 100 fs over 12 decades in time. We find that the photocycle kinetics and structural dynamics of PYP in the crystalline form deviate from those in solution from the very first steps following photon absorption. This illustrates that ultrafast TRC results cannot be uncritically extrapolated to in vivo function, and that comparative spectroscopic experiments on proteins in crystalline and solution states can help identify structural intermediates under native conditions.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X/métodos , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/metabolismo , Proteínas de Bactérias/efeitos da radiação , Cinética , Luz , Estrutura Molecular , Processos Fotoquímicos , Fotorreceptores Microbianos/efeitos da radiação , Conformação Proteica , Análise Espectral
17.
Proc Natl Acad Sci U S A ; 117(33): 19731-19736, 2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32759207

RESUMO

Cyanobacteriochromes are photoreceptors in cyanobacteria that exhibit a wide spectral coverage and unique photophysical properties from the photoinduced isomerization of a linear tetrapyrrole chromophore. Here, we integrate femtosecond-resolved fluorescence and transient-absorption methods and unambiguously showed the significant solvation dynamics occurring at the active site from a few to hundreds of picoseconds. These motions of local water molecules and polar side chains are continuously convoluted with the isomerization reaction, leading to a nonequilibrium processes with continuous active-site motions. By mutations of critical residues at the active site, the modified local structures become looser, resulting in faster solvation relaxations and isomerization reaction. The observation of solvation dynamics is significant and critical to the correct interpretation of often-observed multiphasic dynamic behaviors, and thus the previously invoked ground-state heterogeneity may not be relevant to the excited-state isomerization reaction.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Cianobactérias/metabolismo , Fotorreceptores Microbianos/química , Proteínas de Bactérias/genética , Domínio Catalítico , Cianobactérias/química , Cianobactérias/genética , Isomerismo , Cinética , Fotorreceptores Microbianos/genética , Fotorreceptores Microbianos/metabolismo
18.
ACS Synth Biol ; 9(8): 2076-2086, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32610009

RESUMO

The self-assembly of different cell types into multicellular structures and their organization into spatiotemporally controlled patterns are both challenging and extremely powerful to understand how cells function within tissues and for bottom-up tissue engineering. Here, we not only independently control the self-assembly of two cell types into multicellular architectures with blue and red light, but also achieve their self-sorting into distinct assemblies. This required developing two cell types that form selective and homophilic cell-cell interactions either under blue or red light using photoswitchable proteins as artificial adhesion molecules. The interactions were individually triggerable with different colors of light, reversible in the dark, and provide noninvasive and temporal control over the cell-cell adhesions. In mixtures of the two cells, each cell type self-assembled independently upon orthogonal photoactivation, and cells sorted out into separate assemblies based on specific self-recognition. These self-sorted multicellular architectures provide us with a powerful tool for producing tissue-like structures from multiple cell types and investigate principles that govern them.


Assuntos
Adesão Celular/efeitos da radiação , Luz , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Linhagem Celular Tumoral , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Humanos , Cinética , Optogenética , Fotorreceptores Microbianos/genética , Fotorreceptores Microbianos/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo
19.
Proc Natl Acad Sci U S A ; 117(27): 15573-15580, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571944

RESUMO

Cyanobacteriochromes (CBCRs) are small, bistable linear tetrapyrrole (bilin)-binding light sensors which are typically found as modular components in multidomain cyanobacterial signaling proteins. The CBCR family has been categorized into many lineages that roughly correlate with their spectral diversity, but CBCRs possessing a conserved DXCF motif are found in multiple lineages. DXCF CBCRs typically possess two conserved Cys residues: a first Cys that remains ligated to the bilin chromophore and a second Cys found in the DXCF motif. The second Cys often forms a second thioether linkage, providing a mechanism to sense blue and violet light. DXCF CBCRs have been described with blue/green, blue/orange, blue/teal, and green/teal photocycles, and the molecular basis for some of this spectral diversity has been well established. We here characterize AM1_1499g1, an atypical DXCF CBCR that lacks the second cysteine residue and exhibits an orange/green photocycle. Based on prior studies of CBCR spectral tuning, we have successfully engineered seven AM1_1499g1 variants that exhibit robust yellow/teal, green/teal, blue/teal, orange/yellow, yellow/green, green/green, and blue/green photocycles. The remarkable spectral diversity generated by modification of a single CBCR provides a good template for multiplexing synthetic photobiology systems within the same cellular context, thereby bypassing the time-consuming empirical optimization process needed for multiple probes with different protein scaffolds.


Assuntos
Proteínas de Bactérias/metabolismo , Evolução Molecular , Luz , Fotorreceptores Microbianos/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/efeitos da radiação , Cor , Cianobactérias/genética , Cianobactérias/metabolismo , Cianobactérias/efeitos da radiação , Mutagênese Sítio-Dirigida , Nostoc/genética , Nostoc/metabolismo , Nostoc/efeitos da radiação , Fotobiologia/métodos , Fotorreceptores Microbianos/efeitos da radiação , Biologia Sintética/métodos , Tetrapirróis/metabolismo
20.
Proc Natl Acad Sci U S A ; 117(28): 16356-16362, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32591422

RESUMO

Phytochromes are a diverse family of bilin-binding photoreceptors that regulate a wide range of physiological processes. Their photochemical properties make them attractive for applications in optogenetics and superresolution microscopy. Phytochromes undergo reversible photoconversion triggered by the Z ⇄ E photoisomerization about the double bond in the bilin chromophore. However, it is not fully understood at the molecular level how the protein framework facilitates the complex photoisomerization dynamics. We have studied a single-domain bilin-binding photoreceptor All2699g1 (Nostoc sp. PCC 7120) that exhibits photoconversion between the red light-absorbing (Pr) and far red-absorbing (Pfr) states just like canonical phytochromes. We present the crystal structure and examine the photoisomerization mechanism of the Pr form as well as the formation of the primary photoproduct Lumi-R using time-resolved spectroscopy and hybrid quantum mechanics/molecular mechanics simulations. We show that the unusually long excited state lifetime (broad lifetime distribution centered at ∼300 picoseconds) is due to the interactions between the isomerizing pyrrole ring D and an adjacent conserved Tyr142. The decay kinetics shows a strongly distributed character which is imposed by the nonexponential protein dynamics. Our findings offer a mechanistic insight into how the quantum efficiency of the bilin photoisomerization is tuned by the protein environment, thereby providing a structural framework for engineering bilin-based optical agents for imaging and optogenetics applications.


Assuntos
Fitocromo/química , Fitocromo/metabolismo , Pigmentos Biliares/química , Pigmentos Biliares/metabolismo , Cristalografia por Raios X , Isomerismo , Cinética , Modelos Moleculares , Nostoc/metabolismo , Processos Fotoquímicos , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/metabolismo , Conformação Proteica , Análise Espectral , Relação Estrutura-Atividade
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