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1.
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
2.
Appl Environ Microbiol ; 84(9)2018 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-29475867

RESUMO

In many pro- and eukaryotes, a retinal-based proton pump equips the cell to drive ATP synthesis with (sun)light. Such pumps, therefore, have been proposed as a plug-in for cyanobacteria to artificially increase the efficiency of oxygenic photosynthesis. However, little information on the metabolism of retinal, their chromophore, is available for these organisms. We have studied the in vivo roles of five genes (sll1541, slr1648, slr0091, slr1192, and slr0574) potentially involved in retinal metabolism in Synechocystis sp. strain PCC 6803. With a gene deletion approach, we have shown that Synechocystis apo-carotenoid-15,15-oxygenase (SynACO), encoded by gene sll1541, is an indispensable enzyme for retinal synthesis in Synechocystis, presumably via asymmetric cleavage of ß-apo-carotenal. The second carotenoid oxygenase (SynDiox2), encoded by gene slr1648, competes with SynACO for substrate(s) but only measurably contributes to retinal biosynthesis in stationary phase via an as-yet-unknown mechanism. In vivo degradation of retinal may proceed through spontaneous chemical oxidation and via enzyme-catalyzed processes. Deletion of gene slr0574 (encoding CYP120A1), but not of slr0091 or of slr1192, causes an increase (relative to the level in wild-type Synechocystis) in the retinal content in both the linear and stationary growth phases. These results suggest that CYP120A1 does contribute to retinal degradation. Preliminary data obtained using 13C-labeled retinal suggest that conversion to retinol and retinoic acid and subsequent further oxidation also play a role. Deletion of sll1541 leads to deficiency in retinal synthesis and allows the in vivo reconstitution of far-red-absorbing holo-proteorhodopsin with exogenous retinal analogues, as demonstrated here for all-trans 3,4-dehydroretinal and 3-methylamino-16-nor-1,2,3,4-didehydroretinal.IMPORTANCE Retinal is formed by many cyanobacteria and has a critical role in most forms of life for processes such as photoreception, growth, and stress survival. However, the metabolic pathways in cyanobacteria for synthesis and degradation of retinal are poorly understood. In this paper we identify genes involved in its synthesis, characterize their role, and provide an initial characterization of the pathway of its degradation. This led to the identification of sll1541 (encoding SynACO) as the essential gene for retinal synthesis. Multiple pathways for retinal degradation presumably exist. These results have allowed us to construct a strain that expresses a light-dependent proton pump with an action spectrum extending beyond 700 nm. The availability of this strain will be important for further work aimed at increasing the overall efficiency of oxygenic photosynthesis.


Assuntos
Proteínas de Bactérias/genética , Sequência de Bases , Deleção de Sequência , Synechocystis/genética , Proteínas de Bactérias/biossíntese , Expressão Gênica , Rodopsinas Microbianas , Synechocystis/metabolismo
3.
J Proteomics ; 75(7): 2205-15, 2012 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-22326961

RESUMO

Chemical cross-linking of protein complexes combined with mass spectrometry is a powerful approach to obtain 3-D structural information by revealing amino residues that are in close spatial proximity. To increase the efficiency of mass spectrometric analysis, we have demonstrated the selective enrichment of cross-linked peptides from the 350 kDa protein complex RNA polymerase (RNAP) from Bacillus subtilis. Bis(succinimidyl)-3-azidomethyl glutarate was used as a cross-linker along with an azide-reactive cyclooctyne-conjugated resin to capture target peptides. Subsequently released peptides were fractionated by strong cation exchange chromatography and subjected to LC-MS/MS. We mapped 10 different intersubunit and 24 intrasubunit cross-links by xComb database searching supplied with stringent criteria for confirmation of the proposed structure of candidate cross-linked peptides. The cross-links fit into a homology model of RNAP. Cross-links between ß lobe 1 and the ß' downstream jaw, and cross-links involving the N-terminal and C-terminal parts of the α subunits suggest conformational flexibility. The analytical strategy presented here can be applied to map protein-protein interactions at the amino acid level in biological assemblies of similar complexity. Our approach enables the exploration of alternative peptide fragmentation techniques that may further facilitate cross-link analysis.


Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , RNA Polimerases Dirigidas por DNA/química , Bases de Dados de Proteínas , Modelos Moleculares , Peptídeos/química , Homologia Estrutural de Proteína , Reagentes para Ligações Cruzadas/química , Espectrometria de Massas , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína
4.
FEBS Lett ; 585(1): 167-72, 2011 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-21110976

RESUMO

The redox-midpoint potential of the FAD chromophore in the BLUF domain of anti-transcriptional regulator AppA from Rhodobacter sphaeroides equals ∼-260mV relative to the calomel electrode. Altering the structure of its chromophore-binding pocket through site-directed mutagenesis brings this midpoint potential closer to that of free flavin in aqueous solution. The redox-midpoint potential of this BLUF domain is intermediate between those of LOV domains and Cryptochromes, which may rationalize the primary photochemistry observed in these three flavin-containing photoreceptor families. These results also imply that LOV domains, among the flavin-containing photosensory receptors, are least sensitive to intracellular chemical reduction in the dark.


Assuntos
Proteínas de Bactérias/metabolismo , Flavina-Adenina Dinucleotídeo/metabolismo , Flavoproteínas/metabolismo , Rhodobacter sphaeroides/metabolismo , Algoritmos , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação/genética , Criptocromos/química , Criptocromos/metabolismo , Flavina-Adenina Dinucleotídeo/química , Flavinas/química , Flavinas/metabolismo , Flavoproteínas/química , Flavoproteínas/genética , Cinética , Luz , Mutagênese Sítio-Dirigida , Oxirredução/efeitos dos fármacos , Oxirredução/efeitos da radiação , Oxigênio/farmacologia , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/metabolismo , Rhodobacter sphaeroides/genética , Espectrofotometria
5.
J Phys Chem B ; 113(50): 16251-6, 2009 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-19928893

RESUMO

Proteorhodopsin (pR) is a membrane-embedded proton pump from the microbial rhodopsin family. Light absorption by its retinal chromophore initiates a photocycle, driven by trans/cis isomerization on the femtosecond to picosecond time scales. Here, we report a study on the photoisomerization dynamics of the retinal chromophore of pR, using dispersed ultrafast pump-dump-probe spectroscopy. The application of a pump pulse initiates the photocycle, and with an appropriately tuned dump pulse applied at a time delay after the dump, the molecules in the initial stages of the photochemical process can be de-excited and driven back to the ground state. In this way, we were able to resolve an intermediate on the electronic ground state that represents chromophores that are unsuccessful in isomerization. In particular, the fractions of molecules that undergo slow isomerization (20 ps) have a high probability to enter this state rather than the isomerized K-state. On the ground state reaction surface, return to the stable ground state conformation via a structural or vibrational relaxation occurs in 2-3 ps. Inclusion of this intermediate in the kinetic scheme led to more consistent spectra of the retinal-excited state, and to a more accurate estimation of the quantum yield of isomerization (Phi = 0.4 at pH 6).


Assuntos
Retinaldeído/química , Rodopsina/química , Absorção , Isomerismo , Processos Fotoquímicos , Rodopsinas Microbianas , Espectrofotometria , Fatores de Tempo
6.
Biophys J ; 94(10): 4020-30, 2008 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-18234812

RESUMO

Proteorhodopsin is an ion-translocating member of the microbial rhodopsin family. Light absorption by its retinal chromophore initiates a photocycle, driven by trans/cis isomerization, leading to transmembrane translocation of a proton toward the extracellular side of the cytoplasmic membrane. Here we report a study on the photoisomerization dynamics of the retinal chromophore of proteorhodopsin, using femtosecond time-resolved spectroscopy, by probing in the visible- and in the midinfrared spectral regions. Experiments were performed both at pH 9.5 (a physiologically relevant pH value in which the primary proton acceptor of the protonated Schiff base, Asp(97), is deprotonated) and at pH 6.5 (with Asp(97) protonated). Simultaneous analysis of the data sets recorded in the two spectral regions and at both pH values reveals a multiexponential excited state decay, with time constants of approximately 0.2 ps, approximately 2 ps, and approximately 20 ps. From the difference spectra associated with these dynamics, we conclude that there are two chromophore-isomerization pathways that lead to the K-state: one with an effective rate of approximately (2 ps)(-1) and the other with a rate of approximately (20 ps)(-1). At high pH, both pathways are equally effective, with an estimated quantum yield for K-formation of approximately 0.7. At pH 6.5, the slower pathway is less productive, which results in an isomerization quantum yield of 0.5. We further observe an ultrafast response of residue Asp(227), which forms part of the counterion complex, corresponding to a strengthening of its hydrogen bond with the Schiff base on K-state formation; and a feature that develops on the 0.2 ps and 2 ps timescale and probably reflects a response of an amide II band in reaction to the isomerization process.


Assuntos
Modelos Químicos , Modelos Moleculares , Fotoquímica/métodos , Rodopsina/química , Rodopsina/efeitos da radiação , Análise Espectral/métodos , Simulação por Computador , Cinética , Luz , Fótons , Conformação Proteica/efeitos da radiação , Rodopsina/ultraestrutura , Rodopsinas Microbianas
7.
Photochem Photobiol Sci ; 6(5): 571-9, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17487311

RESUMO

The bacterial photoreceptor protein photoactive yellow protein (PYP) covalently binds the chromophore 4-hydroxy coumaric acid, tuning (spectral) characteristics of this cofactor. Here, we study this binding and tuning using a combination of pointmutations and chromophore analogs. In all photosensor proteins studied to date the covalent linkage of the chromophore to the apoprotein is dispensable for light-induced catalytic activation. We analyzed the functional importance of the covalent linkage using an isosteric chromophore-protein variant in which the cysteine is replaced by a glycine residue and the chromophore by thiomethyl-p-coumaric acid (TMpCA). The model compound TMpCA is shown to weakly complex with the C69G protein. This non-covalent binding results in considerable tuning of both the pKa and the color of the chromophore. The photoactivity of this system, however, was strongly impaired, making PYP the first known photosensor protein in which the covalent linkage of the chromophore is of paramount importance for the functional activity of the protein in vitro. We also studied the influence of chromophore analogs on the color and photocycle of PYP, not only in WT, but especially in the E46Q mutant, to test if effects from both chromophore and protein modifications are additive. When the E46Q protein binds the sinapinic acid chromophore, the color of the protein is effectively changed from yellow to orange. The altered charge distribution in this protein also results in a changed pKa value for chromophore protonation, and a strongly impaired photocycle. Both findings extend our knowledge of the photochemistry of PYP for signal generation.


Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Ácidos Cumáricos/química , Fotorreceptores Microbianos/química , Fotorreceptores Microbianos/metabolismo , Proteínas de Bactérias/genética , Concentração de Íons de Hidrogênio , Fotorreceptores Microbianos/genética , Mutação Puntual , Propionatos , Ligação Proteica , Espectrofotometria Ultravioleta
8.
Biochemistry ; 46(11): 3129-37, 2007 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-17311415

RESUMO

Phototropins are autophosphorylating serine/threonine kinases responsible for blue-light perception in plants; their action gives rise to phototropism, chloroplast relocation, and opening of stomatal guard cells. The kinase domain constitutes the C-terminal part of Avena sativa phototropin 1. The N-terminal part contains two light, oxygen, or voltage (LOV) sensing domains, LOV1 and LOV2; each binds a flavin mononucleotide (FMN) chromophore (lambdamax = 447 nm, termed D447) and forms the light-sensitive domains, of which LOV2 is the principal component. Blue-light absorption produces a covalent adduct between a very conserved nearby cysteine residue and the C(4a) atom of the FMN moiety via the triplet state of the flavin. The covalent adduct thermally decays to regenerate the D447 dark state, with a rate that may vary by several orders of magnitude between different species. We report that the imidazole base can act as a very efficient enhancer of the dark recovery of A. sativa phot1 LOV2 (AsLOV2) and some other well-characterized LOV domains. Imidazole accelerates the thermal decay of AsLOV2 by 3 orders of magnitude in the submolar concentration range, via a base-catalyzed mechanism involving base abstraction of the FMN N(5)-H adduct state and subsequent reprotonation of the reactive cysteine. The LOV2 crystal structure suggests that the imidazole molecules may act from a cavity located in the vicinity of the FMN, explaining its high efficiency, populated through a channel connecting the cavity to the protein surface. Use of pH titration and chemical inactivation by diethyl pyrocarbonate (DEPC) suggests that histidines located at the surface of the LOV domain act as base catalysts via an as yet unidentified H-bond network, operating at a rate of (55 s)-1 at pH 8. In addition, molecular processes other than histidine-mediated base catalysis contibute significantly to the total thermal decay rate of the adduct and operate at a rate constant of (65 s)-1, leading to a net adduct decay time constant of 30 s at pH 8.


Assuntos
Flavoproteínas/fisiologia , Imidazóis/química , Proteínas Serina-Treonina Quinases/fisiologia , Sequência de Aminoácidos , Avena/metabolismo , Escuridão , Dietil Pirocarbonato/química , Mononucleotídeo de Flavina/química , Flavoproteínas/química , Histidina/química , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Fototropismo , Estrutura Terciária de Proteína , Proteínas Serina-Treonina Quinases/química
9.
Microbiology (Reading) ; 148(Pt 1): 69-78, 2002 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-11782500

RESUMO

Intracellular signal transfer in bacteria is dominated by phosphoryl transfer between conserved transmitter and receiver domains in regulatory proteins of so-called two-component systems. Escherichia coli contains 30 such systems, which allow it to modulate gene expression, enzyme activity and the direction of flagellar rotation. The authors have investigated whether, and to what extent, these separate systems form (an) interacting network(s) in vivo, focussing on interactions between four major systems, involved in the responses to the availability of phosphorylated sugars (Uhp), phosphate (Pho), nitrogen (Ntr) and oxygen (Arc). Significant cross-talk was not detectable in wild-type cells. Decreasing expression levels of succinate dehydrogenase (reporting Arc activation), upon activation of the Pho system, appeared to be independent of signalling through PhoR. Cross-talk towards NtrC did occur, however, in a ntrB deletion strain, upon joint activation of Pho, Ntr and Uhp. UhpT expression was demonstrated when cells were grown on pyruvate, through non-cognate phosphorylation of UhpA by acetyl phosphate.


Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Transdução de Sinais , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Nitrogênio/metabolismo , Oxigênio/metabolismo , Fosfatos/metabolismo , Fosforilação
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