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1.
J Am Chem Soc ; 143(46): 19614-19628, 2021 11 24.
Artigo em Inglês | MEDLINE | ID: mdl-34780163

RESUMO

We demonstrate that the Halorhodospira halophila (Hhal) photoactive yellow protein (PYP) is not representative of the greater PYP family. The photodynamics of the PYP isolated from Salinibacter ruber (Srub) is characterized with a comprehensive range of spectroscopic techniques including ultrafast transient absorption, photostationary light titrations, Fourier transform infrared, and cryokinetics spectroscopies. We demonstrate that the dark-adapted pG state consists of two subpopulations differing in the protonation state of the chromophore and that both are photoactive, with the protonated species undergoing excited-state proton transfer. However, the primary I0 photoproduct observed in the Hhal PYP photocycle is absent in the Srub PYP photodynamics, which indicates that this intermediate, while important in Hhal photodynamics, is not a critical intermediate in initiating all PYP photocycles. The excited-state lifetime of Srub PYP is the longest of any PYP resolved to date (∼30 ps), which we ascribe to the more constrained chromophore binding pocket of Srub PYP and the absence of the critical Arg52 residue found in Hhal PYP. The final stage of the Srub PYP photocycle involves the slowest known thermal dark reversion of a PYP (∼40 min vs 350 ms in Hhal PYP). This property allowed the characterization of a pH-dependent equilibrium between the light-adapted pB state with a protonated cis chromophore and a newly resolved pG' intermediate with a deprotonated cis chromophore and pG-like protein conformation. This result demonstates that protein conformational changes and chromophore deprotonation precede chromophore reisomerization during the thermal recovery of the PYP photocycle.


Assuntos
Proteínas de Bactérias/química , Bacteroidetes/química , Halorhodospira halophila/química , Fotorreceptores Microbianos/química , Proteínas de Bactérias/isolamento & purificação , Processos Fotoquímicos , Fotorreceptores Microbianos/isolamento & purificação , Conformação Proteica , Prótons , Estereoisomerismo , Temperatura
2.
Biochemistry ; 60(4): 274-288, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33439010

RESUMO

Cyanobacteriochromes (CBCRs) are cyanobacterial photoreceptors that exhibit photochromism between two states: a thermally stable dark-adapted state and a metastable light-adapted state with bound linear tetrapyrrole (bilin) chromophores possessing 15Z and 15E configurations, respectively. The photodynamics of canonical red/green CBCRs have been extensively studied; however, the time scales of their excited-state lifetimes and subsequent ground-state evolution rates widely differ and, at present, remain difficult to predict. Here, we compare the photodynamics of two closely related red/green CBCRs that have substantial sequence identity (∼68%) and similar chromophore environments: AnPixJg2 from Anabaena sp. PCC 7120 and NpR6012g4 from Nostoc punctiforme. Using broadband transient absorption spectroscopy on the primary (125 fs to 7 ns) and secondary (7 ns to 10 ms) time scales together with global analysis modeling, our studies revealed that AnPixJg2 and NpR6012g4 have comparable quantum yields for initiating the forward (15ZPr → 15EPg) and reverse (15EPg → 15ZPr) reactions, which proceed through monotonic and nonmonotonic mechanisms, respectively. In addition to small discrepancies in the kinetics, the secondary reverse dynamics resolved unique features for each domain: intermediate shunts in NpR6012g4 and a Meta-Gf intermediate red-shifted from the 15ZPr photoproduct in AnPixJg2. Overall, this study supports the conclusion that sequence similarity is a useful criterion for predicting pathways of the light-induced evolution and quantum yield of generating primary intermediate Φp within subfamilies of CBCRs, but more studies are still needed to develop a comprehensive molecular level understanding of these processes.


Assuntos
Anabaena/química , Proteínas de Bactérias/química , Luz , Nostoc/química
3.
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
4.
J Phys Chem Lett ; 11(11): 4245-4252, 2020 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-32374610

RESUMO

The photocycle of a reversible photoisomerizing rhodopsin mimic (M2) is investigated. This system, based on the cellular retinoic acid binding protein, is structurally different from natural rhodopsin systems, but exhibits a similar isomerization upon light irradiation. More specifically, M2 displays a 15-cis to all-trans conversion of retinal protonated Schiff base (rPSB) and all-trans to 15-cis isomerization of unprotonated Schiff base (rUSB). Here we use hybrid quantum mechanics/molecular mechanics (QM/MM) tools coupled with transient absorption and cryokinetic UV-vis spectroscopies to investigate these isomerization processes. The results suggest that primary rPSB photoisomerization of M2 occurs around the C13═C14 double bond within 2 ps following an aborted-bicycle pedal (ABP) isomerization mechanism similar to natural microbial rhodopsins. The rUSB isomerization is much slower and occurs within 48 ps around the C15═N double bond. Our findings reveal the possibility to engineer naturally occurring mechanistic features into artificial rhodopsins and also constitute a step toward understanding the photoisomerization of UV pigments. We conclude by reinforcing the idea that the presence of the retinal chromophore inside a tight protein cavity is not mandatory to exhibit ABP mechanism.


Assuntos
Rodopsina/química , Rodopsina/efeitos da radiação , Isomerismo , Luz , Teoria Quântica , Receptores do Ácido Retinoico , Bases de Schiff/química , Análise Espectral/métodos
5.
Photochem Photobiol Sci ; 18(10): 2539-2552, 2019 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-31528964

RESUMO

Cyanobacteriochromes (CBCRs) are photosensitive proteins that are distantly related to the phytochrome family of photoreceptors and, like phytochromes, exhibit photoactivity initiated by the excited-state photoisomerization of a covalently bound bilin chromophore. The canonical red/green photoswitching sub-family is the most studied class of CBCRs studied to date. Recently, a comparative study of the ultrafast (100 fs-10 ns) forward photodynamics of nine red/green photoswitching CBCR domains isolated from Nostoc punctiforme were reported (S. M. Gottlieb, P. W. Kim, C.-W. Chang, S. J. Hanke, R. J. Hayer, N. C. Rockwell, S. S. Martin, J. C. Lagarias and D. S. Larsen, Conservation and Diversity in the Primary Forward Photodynamics of Red/Green Cyanobacteriochromes, Biochemistry, 2015, 54, 1028-1042). We extend this study by characterizing the secondary (10 ns-1 ms) forward photodynamics of eight red/green photoswitching CBCRs from N. punctiforme with broadband time-resolved absorption spectroscopy. We demonstrate that the dynamics of these representative red/green CBCRs can be separated into two coexisting pathways involving a photoactive pathway that is successful in generating the terminal light-adapted 15EPg population and an unsuccessful pathway that stalls after generating a meta-stable Lumi-Of intermediate. The photoactive pathway evolves through a similar mechanism from excitation of the dark-adapted 15ZPr state to generate a far-red absorbing Lumi-Rf and then via a succession of blue-shifting photointermediates to ultimately generate the 15EPg state. This suggests a steady deviation from planarity of the bilin chromophore during the dynamics. While, the general mechanism for this evolution is conserved among these CBCBs, the timescales of these dynamics deviate significantly. Only half of the characterized CBCRs exhibit the unproductive pathways due to photoexcitation of dark-adapted 15ZPo sub-population that upon photoexcitation generates a meta-stable Lumi-Of intermediate, which eventually decays back to the 15ZPo subpopulation. 15ZPo is ascribed the horizontal Asp657 configuration that disrupts H-bonding with the chromophore in the dark-adapted state; its presence can be identified via enhanced absorption of high-energy tail of the electronic absorption spectrum.


Assuntos
Proteínas de Bactérias/química , Nostoc/metabolismo , Fotorreceptores Microbianos/química , Proteínas de Bactérias/metabolismo , Luz , Fotorreceptores Microbianos/metabolismo , Estrutura Terciária de Proteína , Espectrofotometria
6.
Biochemistry ; 58(18): 2297-2306, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-30973006

RESUMO

Cyanobacteriochromes (CBCRs) make up a diverse family of cyanobacterial photoreceptors distantly related to the phytochrome photoreceptors of land plants. At least two lineages of CBCRs have reacquired red-absorbing dark states similar to the phytochrome Pr resting state but are coupled to green-absorbing light-adapted states rather than the canonical far-red-absorbing light-adapted state. One such lineage includes the canonical red/green (R/G) CBCRs that includes AnPixJg2 (UniProtKB Q8YXY7 ) and NpR6012g4 (UniProtKB B2IU14 ) that have been extensively characterized. Here we examine the forward Pr photodynamics of NpR3784 (UniProtKB B2J457 ), a representative member of the second R/G CBCR subfamily. Using broadband transient absorption pump-probe spectroscopy, we characterize both primary (100 fs to 10 ns) and secondary (10 ns to 1 ms) forward (Pr → Pg) photodynamics and compare the results to temperature-jump cryokinetics measurements. Our studies show that primary isomerization dynamics occur on an ∼10 ps timescale, yet remarkably, the red-shifted primary Lumi-Rf photoproduct found in all photoactive canonical R/G CBCRs examined to date is extremely short-lived in NpR3784. These results demonstrate that differences in reaction pathways reflect the evolutionary history of R/G CBCRs despite the convergent evolution of their photocycle end products.


Assuntos
Proteínas de Bactérias/metabolismo , Luz , Nostoc/metabolismo , Fotorreceptores Microbianos/metabolismo , Cinética , Nostoc/efeitos da radiação , Processos Fotoquímicos/efeitos da radiação , Fotorreceptores Microbianos/efeitos da radiação , Espectrofotometria
7.
Biochemistry ; 58(18): 2307-2317, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-30977638

RESUMO

In the companion paper (10.1021/acs.biochem.8b01274), we examined the forward Pr photodynamics of NpR3784 (UniProtKB B2J457 ), a representative member of a noncanonical red/green (R/G) cyanobacteriochrome (CBCR) subfamily. Here the reverse Pg → Pr photodynamics of NpR3784 was studied by broadband transient absorption pump-probe spectroscopy. Primary (100 fs to 10 ns) and secondary (10 ns to 1 ms) photodynamics were characterized over nine decades of time, which also were complemented with temperature-jump cryokinetics measurements. In contrast with canonical R/G CBCRs, the NpR3784 reverse photoconversion yielded two spectrally distinct primary photoproducts, Lumi-Go and Lumi-Gr, which decay on different time scales. The two primary photoproducts of NpR3784 equilibrate on the 40 ns time scale and subsequently propagate as a single intermediate population into Pr. Such heterogeneity could arise from differences in the direction of D-ring rotation, in chromophore protonation or hydrogen bonding, or in the mobility of protein residues or of solvent water nearby the chromophore or some combination therein. We conclude that the atypical photodynamics of NpR3784 reflects chromophore-protein interactions that differ from those present in the canonical R/G CBCR family.


Assuntos
Proteínas de Bactérias/metabolismo , Luz , Nostoc/metabolismo , Fotorreceptores Microbianos/metabolismo , Cinética , Nostoc/efeitos da radiação , Processos Fotoquímicos/efeitos da radiação , Fotorreceptores Microbianos/efeitos da radiação , Espectrofotometria
8.
J Phys Chem Lett ; 9(18): 5351-5357, 2018 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-30157382

RESUMO

The optical control of spin state is of interest in the development of spintronic materials for data processing and storage technologies. Photomagnetic effects at the single-molecule level have recently been observed in the thin film state at 300 K in photochromic cobalt dioxolenes. Visible light excitation leads to ring-closure of a photochromic spirooxazine bound to a cobalt dioxolene, which leads to generation of a high magnetization state. Formation of the photomagnetic state occurs through a photoisomerization-induced spin-charge excited-state process and is dictated by the spirooxazine ligand dynamics. Here, we report a mechanistic investigation by ultrafast spectroscopy in the UV-vis region of the photochemical ring-closing process in the parent spirooxazine, azahomoadamantylphenanthroline spirooxazine, and the photomagnetic spirooxazine cobalt-dioxolene complex. The cobalt appears to stabilize a photomerocycanine transient intermediate, presumably the TCC isomer, formed along the ground-state potential energy surface (PES). Structural changes associated with the TCC isomer induces formation of the high-spin Co(II) form, suggesting that magnetization dymanics can occur along the excited-state PES, leading to ultrafast switching on the ps time scale. We demonstrate the full ring closure of the spiro-oxazine ligand is not required to switch magnetization states which can be induced with a higher yielding isomerization reaction. The ability of this system to undergo optically induced spin state switching on the ps time scale in the solid state makes it a promising canididate for resistive nonvolatile memory technologies.

9.
J Phys Chem Lett ; 9(12): 3454-3462, 2018 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-29874080

RESUMO

Phytochrome proteins utilize ultrafast photoisomerization of a linear tetrapyrrole chromophore to detect the ratio of red to far-red light. Femtosecond photodynamics in the PAS-GAF-PHY photosensory core of the Cph1 phytochrome from Synechocystis sp. PCC6803 (Cph1Δ) were resolved with a dual-excitation-wavelength-interleaved pump-probe (DEWI) approach with two excitation wavelengths (600 and 660 nm) at three pH values (6.5, 8.0, and 9.0). Observed spectral and kinetic heterogeneity in the excited-state dynamics were described with a self-consistent model comprised of three spectrally distinct populations with different protonation states (Pr-I, Pr-II, and Pr-III), each composed of multiple kinetically distinct subpopulations. Apparent partitioning among these populations is dictated by pH, temperature, and excitation wavelength. Our studies provide insight into photocycle initiation dynamics at physiological temperatures, implicate the low-pH/low-temperature Pr-I state as the photoactive state in vitro, and implicate an internal hydrogen-bonding network in regulating the photochemical quantum yield.

10.
Biochemistry ; 57(18): 2636-2648, 2018 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-29633829

RESUMO

Forward and reverse primary (<10 ns) and secondary (>10 ns) photodynamics of cyanobacteriochrome (CBCR) NpF2164g7 were characterized by global analysis of ultrafast broadband transient absorption measurements. NpF2164g7 is the most C-terminal bilin-binding GAF domain in the Nostoc punctiforme phototaxis sensor PtxD (locus Npun_F2164). Although a member of the canonical red/green CBCR subfamily phylogenetically, NpF2164g7 exhibits an orange-absorbing 15ZPo dark-adapted state instead of the typical red-absorbing 15ZPr dark-adapted state characteristic of this subfamily. The green-absorbing 15EPg photoproduct of NpF2164g7 is unstable, allowing this CBCR domain to function as a power sensor. Photoexcitation of the 15ZPo state triggers inhomogeneous excited-state dynamics with three spectrally and temporally distinguishable pathways to generate the light-adapted 15EPg state in high yield (estimated at 25-30%). Although observed in other CBCR domains, the inhomogeneity in NpF2164g7 extends far into secondary relaxation dynamics (10 ns -1 ms) through to formation of 15EPg. In the reverse direction, the primary dynamics after photoexcitation of 15EPg are qualitatively similar to those of other red/green CBCRs, but secondary dynamics involve a "pre-equilibrium" step before regenerating 15ZPo. The anomalous photodynamics of NpF2164g7 may reflect an evolutionary adaptation of CBCR sensors that function as broadband light intensity sensors.


Assuntos
Proteínas de Bactérias/química , Cianobactérias/química , Nostoc/química , Fotorreceptores Microbianos/química , Cinética , Luz , Processos Fotoquímicos , Fototaxia/efeitos dos fármacos , Fototaxia/efeitos da radiação
11.
Proc Natl Acad Sci U S A ; 115(17): 4387-4392, 2018 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-29632180

RESUMO

Phytochrome photoreceptors control plant growth, development, and the shade avoidance response that limits crop yield in high-density agricultural plantings. Cyanobacteriochromes (CBCRs) are distantly related photosensory proteins that control cyanobacterial metabolism and behavior in response to light. Photoreceptors in both families reversibly photoconvert between two photostates via photoisomerization of linear tetrapyrrole (bilin) chromophores. Spectroscopic and biochemical studies have demonstrated heterogeneity in both photostates, but the structural basis for such heterogeneity remains unclear. We report solution NMR structures for both photostates of the red/green CBCR NpR6012g4 from Nostoc punctiforme In addition to identifying structural changes accompanying photoconversion, these structures reveal structural heterogeneity for residues Trp655 and Asp657 in the red-absorbing NpR6012g4 dark state, yielding two distinct environments for the phycocyanobilin chromophore. We use site-directed mutagenesis and fluorescence and absorbance spectroscopy to assign an orange-absorbing population in the NpR6012g4 dark state to the minority configuration for Asp657. This population does not undergo full, productive photoconversion, as shown by time-resolved spectroscopy and absorption spectroscopy at cryogenic temperature. Our studies thus elucidate the spectral and photochemical consequences of structural heterogeneity in a member of the phytochrome superfamily, insights that should inform efforts to improve photochemical or fluorescence quantum yields in the phytochrome superfamily.


Assuntos
Proteínas de Bactérias/química , Nostoc/química , Fitocromo/química , Proteínas de Bactérias/genética , Mutagênese Sítio-Dirigida , Nostoc/genética , Fitocromo/genética , Domínios Proteicos
12.
Annu Rev Biophys ; 47: 291-313, 2018 May 20.
Artigo em Inglês | MEDLINE | ID: mdl-29539272

RESUMO

The first stage in biological signaling is based on changes in the functional state of a receptor protein triggered by interaction of the receptor with its ligand(s). The light-triggered nature of photoreceptors allows studies on the mechanism of such changes in receptor proteins using a wide range of biophysical methods and with superb time resolution. Here, we critically evaluate current understanding of proton and electron transfer in photosensory proteins and their involvement both in primary photochemistry and subsequent processes that lead to the formation of the signaling state. An insight emerging from multiple families of photoreceptors is that ultrafast primary photochemistry is followed by slower proton transfer steps that contribute to triggering large protein conformational changes during signaling state formation. We discuss themes and principles for light sensing shared by the six photoreceptor families: rhodopsins, phytochromes, photoactive yellow proteins, light-oxygen-voltage proteins, blue-light sensors using flavin, and cryptochromes.

13.
Biochemistry ; 57(11): 1733-1747, 2018 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-29465990

RESUMO

Photoactive yellow proteins (PYPs) make up a diverse class of blue-light-absorbing bacterial photoreceptors. Electronic excitation of the p-coumaric acid chromophore covalently bound within PYP results in triphasic quenching kinetics; however, the molecular basis of this behavior remains unresolved. Here we explore this question by examining the excitation-wavelength dependence of the photodynamics of the PYP from Halorhodospira halophila via a combined experimental and computational approach. The fluorescence quantum yield, steady-state fluorescence emission maximum, and cryotrapping spectra are demonstrated to depend on excitation wavelength. We also compare the femtosecond photodynamics in PYP at two excitation wavelengths (435 and 475 nm) with a dual-excitation-wavelength-interleaved pump-probe technique. Multicompartment global analysis of these data demonstrates that the excited-state photochemistry of PYP depends subtly, but convincingly, on excitation wavelength with similar kinetics with distinctly different spectral features, including a shifted ground-state beach and altered stimulated emission oscillator strengths and peak positions. Three models involving multiple excited states, vibrationally enhanced barrier crossing, and inhomogeneity are proposed to interpret the observed excitation-wavelength dependence of the data. Conformational heterogeneity was identified as the most probable model, which was supported with molecular mechanics simulations that identified two levels of inhomogeneity involving the orientation of the R52 residue and different hydrogen bonding networks with the p-coumaric acid chromophore. Quantum calculations were used to confirm that these inhomogeneities track to altered spectral properties consistent with the experimental results.


Assuntos
Proteínas de Bactérias/química , Halorhodospira halophila/química , Luz , Simulação de Dinâmica Molecular , Fotorreceptores Microbianos/química , Proteínas de Bactérias/genética , Halorhodospira halophila/genética , Ligação de Hidrogênio , Fotorreceptores Microbianos/genética , Relação Estrutura-Atividade
14.
Biochemistry ; 57(6): 978-990, 2018 Feb 13.
Artigo em Inglês | MEDLINE | ID: mdl-29303562

RESUMO

Photoinduced charge-transfer dynamics and the influence of cluster size on the dynamics were investigated using five iron-sulfur clusters: the 1Fe-4S cluster in Pyrococcus furiosus rubredoxin, the 2Fe-2S cluster in Pseudomonas putida putidaredoxin, the 4Fe-4S cluster in nitrogenase iron protein, and the 8Fe-7S P-cluster and the 7Fe-9S-1Mo FeMo cofactor in nitrogenase MoFe protein. Laser excitation promotes the iron-sulfur clusters to excited electronic states that relax to lower states. The electronic relaxation lifetimes of the 1Fe-4S, 8Fe-7S, and 7Fe-9S-1Mo clusters are on the picosecond time scale, although the dynamics of the MoFe protein is a mixture of the dynamics of the latter two clusters. The lifetimes of the 2Fe-2S and 4Fe-4S clusters, however, extend to several nanoseconds. A competition between reorganization energies and the density of electronic states (thus electronic coupling between states) mediates the charge-transfer lifetimes, with the 2Fe-2S cluster of Pdx and the 4Fe-4S cluster of Fe protein lying at the optimum leading to them having significantly longer lifetimes. Their long lifetimes make them the optimal candidates for long-range electron transfer and as external photosensitizers for other photoactivated chemical reactions like solar hydrogen production. Potential electron-transfer and hole-transfer pathways that possibly facilitate these charge transfers are proposed.


Assuntos
Bactérias/química , Proteínas de Bactérias/química , Proteínas Ferro-Enxofre/química , Azotobacter vinelandii/química , Domínio Catalítico , Transporte de Elétrons , Ferredoxinas/química , Modelos Moleculares , Oxirredução , Oxirredutases/química , Conformação Proteica , Pseudomonas putida/química , Pyrococcus furiosus/química , Rubredoxinas/química
15.
J Phys Chem Lett ; 8(18): 4498-4503, 2017 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-28872878

RESUMO

Iron-sulfur proteins play essential roles in various biological processes. Their electronic structure and vibrational dynamics are key to their rich chemistry but nontrivial to unravel. Here, the first ultrafast transient absorption and impulsive coherent vibrational spectroscopic (ICVS) studies on 2Fe-2S clusters in Rhodobacter capsulatus ferreodoxin VI are characterized. Photoexcitation initiated populations on multiple excited electronic states that evolve into each other in a long-lived charge-transfer state. This suggests a potential light-induced electron-transfer pathway as well as the possibility of using iron-sulfur proteins as photosensitizers for light-dependent enzymes. A tyrosine chain near the active site suggests potential hole-transfer pathways and affirms this electron-transfer pathway. The ICVS data revealed vibrational bands at 417 and 484 cm-1, with the latter attributed to an excited-state mode. The temperature dependence of the ICVS modes suggests that the temperature effect on protein structure or conformational heterogeneities needs to be considered during cryogenic temperature studies.


Assuntos
Proteínas Ferro-Enxofre/química , Conformação Proteica , Rhodobacter capsulatus/fisiologia , Temperatura , Espectroscopia de Ressonância de Spin Eletrônica , Ferredoxinas , Guanina/análogos & derivados , Proteínas Ferro-Enxofre/fisiologia , Oxirredução , Fotoquímica , Análise Espectral , Enxofre/química , Vibração
16.
J Phys Chem Lett ; 7(24): 5212-5218, 2016 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-27973895

RESUMO

The photoactive yellow protein (PYP) from Halorhodospira halophila (Hhal) is a bacterial photoreceptor and model system for exploring functional protein dynamics. We report ultrafast spectroscopy experiments that probe photocycle initiation dynamics in the PYP domain from the multidomain PYP-phytochrome-related photoreceptor from Rhodospirillum centenum (Rcen). As with Hhal PYP, Rcen PYP exhibits similar excited-state dynamics; in contrast, Rcen PYP exhibits altered photoproduct ground-state dynamics in which the primary I0 intermediate as observed in Hhal PYP is absent. This property is attributed to a tighter, more sterically constrained binding pocket around the p-coumaric acid chromophore due to a change in the Rcen PYP protein structure that places Phe98 instead of Met100 in contact with the chromophore. Hence, the I0 state is not a necessary step for the initiation of productive PYP photocycles and the ubiquitously studied Hhal PYP may not be representative of the broader PYP family of photodynamics.


Assuntos
Proteínas de Bactérias/química , Halorhodospira halophila , Fotorreceptores Microbianos/química , Análise Espectral , Ácidos Cumáricos , Fitocromo/metabolismo , Propionatos
17.
Biochemistry ; 55(44): 6138-6149, 2016 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-27749038

RESUMO

We explored the photoisomerization mechanisms in novel homologues of photoactive yellow protein (PYP) from Leptospira biflexa (Lbif) to identify conserved features and functional diversity in the primary photochemistry of this family of photoreceptors. In close agreement with the prototypical PYP from Halorhodospira halophila (Hhal), we observe excited-state absorbance near 375 nm and stimulated emission near 500 nm, with triphasic excited-state decay. While the excited-state decay for Lbif PYP is the slowest among those of known PYPs due to the redistribution of the amplitudes of the three decay components, the quantum yield for productive photocycle entry is very similar to that of Hhal PYP. Pro68 is highly conserved in PYPs and is important for the high photochemical quantum yield in Hhal PYP, but this residue is Ile in wild-type Lbif PYP. The level of photoproduct formation is slightly increased in I68P Lbif PYP, indicating that this residue regulates the photochemical quantum yield in the entire PYP family. Lbif PYP also exhibited a blue-shifted photoproduct previously undiscovered in ultrafast studies of PYP, which we have named pUV. We posit that pUV is a detour in the PYP photocycle with a twisted protonated pCAH configuration. Cryokinetic experiments with Hhal PYP confirmed the presence of pUV, but the population of this state in room-temperature ultrafast experiments is very small. These results resolve the long-standing inconsistency in the literature regarding the existence of a bifurcation in the room-temperature photocycle of PYP.


Assuntos
Proteínas de Bactérias/química , Halorhodospira halophila/química , Leptospira/química , Fotorreceptores Microbianos/química , Ligação de Hidrogênio , Espectrofotometria Ultravioleta
18.
Proc Natl Acad Sci U S A ; 112(7): E776-85, 2015 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-25653339

RESUMO

Mammalian neurotransmitter-gated receptors can be conjugated to photoswitchable tethered ligands (PTLs) to enable photoactivation, or photoantagonism, while preserving normal function at neuronal synapses. "MAG" PTLs for ionotropic and metabotropic glutamate receptors (GluRs) are based on an azobenzene photoswitch that is optimally switched into the liganding state by blue or near-UV light, wavelengths that penetrate poorly into the brain. To facilitate deep-tissue photoactivation with near-infrared light, we measured the efficacy of two-photon (2P) excitation for two MAG molecules using nonlinear spectroscopy. Based on quantitative characterization, we find a recently designed second generation PTL, L-MAG0460, to have a favorable 2P absorbance peak at 850 nm, enabling efficient 2P activation of the GluK2 kainate receptor, LiGluR. We also achieve 2P photoactivation of a metabotropic receptor, LimGluR3, with a new mGluR-specific PTL, D-MAG0460. 2P photoswitching is efficiently achieved using digital holography to shape illumination over single somata of cultured neurons. Simultaneous Ca(2+)-imaging reports on 2P photoswitching in multiple cells with high temporal resolution. The combination of electrophysiology or Ca(2+) imaging with 2P activation by optical wavefront shaping should make second generation PTL-controlled receptors suitable for studies of intact neural circuits.


Assuntos
Compostos Azo/química , Optogenética , Fótons , Receptores de Glutamato/química , Animais , Células Cultivadas , Isomerismo , Neurônios/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Glutamato/metabolismo , Receptores de Glutamato/efeitos da radiação , Análise Espectral/métodos
19.
Biochemistry ; 54(4): 1028-42, 2015 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-25545467

RESUMO

Phytochromes are red/far-red photosensory proteins that detect the ratio of red to far-red light. Crucial to light regulation of plant developmental biology, phytochromes are also found in fungi, bacteria, and eukaryotic algae. In addition to phytochromes, cyanobacteria also can contain distantly related cyanobacteriochromes (CBCRs) that, like phytochromes, utilize the photoisomerization of a linear tetrapyrrole (bilin) chromophore to convert between two photostates with distinct spectral properties. CBCRs exhibit a wide range of photostates spanning the visible and even near-ultraviolet spectrum. In both phytochromes and CBCRs, biosynthesis initially yields a holoprotein with bilin in the 15Z configuration, and the 15E photoproduct can often revert to the 15Z photostate in the absence of light (dark reversion). One CBCR subfamily, red/green CBCRs, typically exhibits red-absorbing dark states and green-absorbing photoproducts. Dark reversion is extremely variable in red/green CBCRs with known examples ranging from seconds to days. One red/green CBCR, NpR6012g4 from Nostoc punctiforme, is also known to exhibit forward photoconversion that has an unusually high quantum yield at ∼40% compared to 10-20% for phytochromes and CBCRs from other subfamilies. In the current study, we use time-resolved pump-probe absorption spectroscopy with broadband detection and multicomponent global analysis to characterize forward photoconversion of seven additional red/green CBCRs from N. punctiforme on an ultrafast time scale. Our results reveal that red/green CBCRs exhibit a conserved pathway for primary forward photoconversion but that considerable diversity exists in their excited-state lifetimes, photochemical quantum yields, and primary photoproduct stabilities.


Assuntos
Proteínas de Bactérias/fisiologia , Cianobactérias/fisiologia , Estimulação Luminosa , Fotorreceptores Microbianos/fisiologia , Fitocromo/fisiologia , Proteínas de Bactérias/química , Estimulação Luminosa/métodos , Fitocromo/química
20.
Appl Spectrosc ; 68(9): 949-54, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25226248

RESUMO

We characterize the femtosecond laser-induced breakdown spectroscopy (fsLIBS) signal for biological tissues as a function of different excitation parameters with femtosecond laser systems. These parameters include laser energy, depth of focus, and number of pulses per focal volume. We used femtosecond laser pulses of 800 nm and energy between 25 and 123 µJ to generate LIBS signals in biological tissues. As expected, we observed a linear increase in the fsLIBS intensity as a function of the laser energy. In addition, we show that moving the beam out of focus and the presence of overlapping pulses on the same focal area leads to a decrease in fsLIBS intensity due to changes in focal spot size. We also demonstrate that fsLIBS can distinguish between different biological tissue samples.


Assuntos
Análise Espectral/métodos , Animais , Osso e Ossos/química , Cartilagem/química , Bovinos , Galinhas , Casca de Ovo/química , Lasers , Microscopia
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