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Klaas J. Hellingwerf - One of the best experts on this subject based on the ideXlab platform.
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FEATURE ARTICLE Photoactive Yellow Protein, A New Type of Photoreceptor Protein: Will This “Yellow Lab” Bring Us Where We Want to Go?|
2016Co-Authors: Klaas J. Hellingwerf, Johnny Hendriks, Thomas GenschAbstract:Photoactive Yellow Protein ( PYP), discovered almost 20 years ago in Ectothiorhodospira (Halorhodospira) Halophila,1 is a 4-hydroxycinnamic acid-containing protein that functions as a blue-light photoreceptor in a behavioral avoidance response in this organism. During the past 10 years, PYP has become a model system for studies in photochemistry and protein folding, to the extent that it has become competitive with the rhodopsins. This is because PYP is small and very water-soluble, forms crystals readily (diffracting to high resolution), and shows excellent chemical- and photo-stability. These overall characteristics have allowed the application of an array of physicochemical techniques to analyze the biological function of PYP, i.e., the translation of a change of the configuration of its 4-hydroxycinnamic acid chromophore into an altered conformation of the surrounding protein. This has led to detailed insight into this process, both temporally and spatially, with respect to the structure of the transient intermediates involved, although we are still quite far from being able to track the position of all atoms in space, upon light activation of the protein in the relevant time domain. Nevertheless, the data already obtained may function as a calibration set in future work, to extend the time span of molecular dynamics simulations of conformational transitions in proteins to the time scale relevant for catalytic turnover. Occasionally, the application of multiple biophysical techniques has led to (seemingly) conflicting results. In one example, this has revealed the fact that the light-induce
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Prokaryotic phototaxis.
Methods in molecular biology (Clifton N.J.), 2009Co-Authors: Wouter D Hoff, Michael A Van Der Horst, Clara B Nudel, Klaas J. HellingwerfAbstract:Microorganisms have various mechanisms at their disposal to react to (changes in) their ambient light climate (i.e., intensity, color, direction, and degree of polarization). Of these, one of the best studied mechanisms is the process of phototaxis. This process can be described as a behavioral migration-response of an organism toward a change in illumination regime. In this chapter we discuss three of these migration responses, based on swimming, swarming, and twitching motility, respectively. Swimming motility has been studied using a wide range of techniques, usually microscopy based. We present a detailed description of the assays used to study phototaxis in liquid cultures of the phototrophic organisms Halobacterium salinarum, Halorhodospira Halophila, and Rhodobacter sphaeroides and briefly describe the molecular basis of these responses. Swarming and twitching motility are processes taking place at the interface between a solid phase and a liquid or gas phase. Although assays to study these processes are relatively straightforward, they are accompanied by technical complications, which we describe. Furthermore, we discuss the molecular processes underlying these forms of motility in Rhodocista centenaria and Synechocystis PCC6803. Recently, it has become clear that also chemotrophic organisms contain photoreceptor proteins that allow them to respond to their ambient light climate. Surprisingly, light-modulated motility responses can also be observed in the chemotrophic organisms Escherichia coli and Acinetobacter calcoaceticus. In the light-modulated surface migration not only "che-like" signal transduction reactions may play a role, but in addition processes as modulation of gene expression and even intermediary metabolism.
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ultrafast infrared spectroscopy reveals a key step for successful entry into the photocycle for photoactive yellow protein
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: L J G W Van Wilderen, Ivo H M Van Stokkum, Klaas J. Hellingwerf, M. A. Van Der Horst, R. Van Grondelle, Marie Louise GrootAbstract:Photoactive proteins such as PYP (photoactive yellow protein) are generally accepted as model systems for studying protein signal state formation. PYP is a blue-light sensor from the bacterium Halorhodospira Halophila . The formation of PYP9s signaling state is initiated by trans-cis isomerization of the p -coumaric acid chromophore upon the absorption of light. The quantum yield of signaling state formation is ≈0.3. Using femtosecond visible pump/mid-IR probe spectroscopy, we investigated the structure of the very short-lived ground state intermediate (GSI) that results from an unsuccessful attempt to enter the photocycle. This intermediate and the first stable GSI on pathway into the photocycle, I 0 , both have a mid-IR difference spectrum that is characteristic of a cis isomer, but only the I 0 intermediate has a chromophore with a broken hydrogen bond with the backbone N atom of Cys-69. We suggest, therefore, that breaking this hydrogen bond is decisive for a successful entry into the photocycle. The chromophore also engages in a hydrogen-bonding network by means of its phenolate group with residues Tyr-42 and Glu-46. We have investigated the role of this hydrogen bond by exchanging the H bond-donating residue Glu-46 with the weaker H bond-donating glutamine (i.e., Gln-46). We have observed that this mutant exhibits virtually identical kinetics and product yields as WT PYP, even though during the I 0 -to-I 1 transition, on the 800-ps time scale, the hydrogen bond of the chromophore with Gln-46 is broken, whereas this hydrogen bond remains intact with Glu-46.
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investigations of the primary events in a bacterial photoreceptor for photomotility photoactive yellow protein pyp
New Journal of Chemistry, 2005Co-Authors: Pascale Changenetbarret, Klaas J. Hellingwerf, Agathe Espagne, Pascal Plaza, Monique M MartinAbstract:PYP, the Photoactive Yellow Protein, is a small water-soluble protein extracted from the cytosol of the halophilic purple bacterium Halorhodospira Halophila. PYP is thought to mediate the phototactic response of the bacterium against blue light. Its chromophore is the deprotonated trans-p-hydroxycinnamic acid covalently linked, via a thioester bond, to the unique cysteine residue of the protein. Upon blue-light irradiation, PYP undergoes a photocycle. As for rhodopsins, the trans to cis isomerization of the chromophore was shown to be the first overall step of this photocycle. From time-resolved spectroscopy measurements on native PYP in solution, it emerged that the reaction involves a series of fast events on the subpicosecond and picosecond timescales, but the reaction path that leads to the formation of the cis isomer is not clear yet. A few years ago, we initiated a comparative study of native PYP and several chromophore analogues in solution in order to try to further clarify the early steps of the photocycle. Our experimental approach consists in probing, in real-time, the ultrafast photoinduced events by transient absorption and gain spectroscopy using the pump–probe technique. In the present paper, we review our experimental results and discuss them within the context of the recent literature.
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Incoherent manipulation of the photoactive yellow protein photocycle with dispersed pump-dump-probe spectroscopy
Biophysical Journal, 2004Co-Authors: Delmar S Larsen, Michael A. Van Der Horst, Ivo H M Van Stokkum, Frank L De Weerd, Mikas Vengris, Klaas J. Hellingwerf, Rienk Van GrondelleAbstract:Photoactive yellow protein is the protein responsible for initiating the "blue-light vision" of Halorhodospira Halophila. The dynamical processes responsible for triggering the photoactive yellow protein photocycle have been disentangled with the use of a novel application of dispersed ultrafast pump-dump-probe spectroscopy, where the photocycle can be started and interrupted with appropriately tuned and timed laser pulses. This "incoherent" manipulation of the photocycle allows for the detailed spectroscopic investigation of the underlying photocycle dynamics and the construction of a fully self-consistent dynamical model. This model requires three kinetically distinct excited-state intermediates, two (ground-state) photocycle intermediates, I0and pR, and a ground-state intermediate through which the protein, after unsuccessful attempts at initiating the photocycle, returns to the equilibrium ground state. Also observed is a previously unknown two-photon ionization channel that generates a radical and an ejected electron into the protein environment. This second excitation pathway evolves simultaneously with the pathway containing the one-photon photocycle intermediates.
Ivo H M Van Stokkum - One of the best experts on this subject based on the ideXlab platform.
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Excitation-Wavelength-Dependent Photocycle Initiation Dynamics Resolve Heterogeneity in the Photoactive Yellow Protein from Halorhodospira Halophila
2018Co-Authors: Tyler L Mix, Ivo H M Van Stokkum, Masato Kumauchi, Elizabeth C Carroll, Dmitry Morozov, Jie Pan, Wendy R Gordon, Andrew Philip, Jack Fuzell, Gerrit GroenhofAbstract: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
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Subpicosecond Excited-State Proton Transfer Preceding Isomerization During the Photorecovery of Photoactive Yellow Protein
2015Co-Authors: Elizabeth C Carroll, Ivo H M Van Stokkum, Wouter D Hoff, Masato Kumauchi, Sang-hun Song, Askat Jailaubekov, Delmar S LarsenAbstract:The ultrafast excited-state dynamics underlying the receptor state photorecovery is resolved in the M100A mutant of the photoactive yellow protein (PYP) from Halorhodospira Halophila. The M100A PYP mutant, with its distinctly slower photocycle than wt PYP, allows isolation of the pB signaling state for study of the photodynamics of the protonated chromophore cis-p-coumaric acid. Transient absorption signals indicate a subpicosecond excited-state proton-transfer reaction in the pB state that results in chromophore deprotonation prior to the cis−trans isomerization required in the photorecovery dynamics of the pG state. Two terminal photoproducts are observed, a blue-absorbing species presumed to be deprotonated trans-p-coumaric acid and an ultraviolet-absorbing protonated photoproduct. These two photoproducts are hypothesized to originate from an equilibrium of open and closed folded forms of the signaling state, I2 and I2′
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ultrafast infrared spectroscopy reveals a key step for successful entry into the photocycle for photoactive yellow protein
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: L J G W Van Wilderen, Ivo H M Van Stokkum, Klaas J. Hellingwerf, M. A. Van Der Horst, R. Van Grondelle, Marie Louise GrootAbstract:Photoactive proteins such as PYP (photoactive yellow protein) are generally accepted as model systems for studying protein signal state formation. PYP is a blue-light sensor from the bacterium Halorhodospira Halophila . The formation of PYP9s signaling state is initiated by trans-cis isomerization of the p -coumaric acid chromophore upon the absorption of light. The quantum yield of signaling state formation is ≈0.3. Using femtosecond visible pump/mid-IR probe spectroscopy, we investigated the structure of the very short-lived ground state intermediate (GSI) that results from an unsuccessful attempt to enter the photocycle. This intermediate and the first stable GSI on pathway into the photocycle, I 0 , both have a mid-IR difference spectrum that is characteristic of a cis isomer, but only the I 0 intermediate has a chromophore with a broken hydrogen bond with the backbone N atom of Cys-69. We suggest, therefore, that breaking this hydrogen bond is decisive for a successful entry into the photocycle. The chromophore also engages in a hydrogen-bonding network by means of its phenolate group with residues Tyr-42 and Glu-46. We have investigated the role of this hydrogen bond by exchanging the H bond-donating residue Glu-46 with the weaker H bond-donating glutamine (i.e., Gln-46). We have observed that this mutant exhibits virtually identical kinetics and product yields as WT PYP, even though during the I 0 -to-I 1 transition, on the 800-ps time scale, the hydrogen bond of the chromophore with Gln-46 is broken, whereas this hydrogen bond remains intact with Glu-46.
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Incoherent manipulation of the photoactive yellow protein photocycle with dispersed pump-dump-probe spectroscopy
Biophysical Journal, 2004Co-Authors: Delmar S Larsen, Michael A. Van Der Horst, Ivo H M Van Stokkum, Frank L De Weerd, Mikas Vengris, Klaas J. Hellingwerf, Rienk Van GrondelleAbstract:Photoactive yellow protein is the protein responsible for initiating the "blue-light vision" of Halorhodospira Halophila. The dynamical processes responsible for triggering the photoactive yellow protein photocycle have been disentangled with the use of a novel application of dispersed ultrafast pump-dump-probe spectroscopy, where the photocycle can be started and interrupted with appropriately tuned and timed laser pulses. This "incoherent" manipulation of the photocycle allows for the detailed spectroscopic investigation of the underlying photocycle dynamics and the construction of a fully self-consistent dynamical model. This model requires three kinetically distinct excited-state intermediates, two (ground-state) photocycle intermediates, I0and pR, and a ground-state intermediate through which the protein, after unsuccessful attempts at initiating the photocycle, returns to the equilibrium ground state. Also observed is a previously unknown two-photon ionization channel that generates a radical and an ejected electron into the protein environment. This second excitation pathway evolves simultaneously with the pathway containing the one-photon photocycle intermediates.
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initial steps of signal generation in photoactive yellow protein revealed with femtosecond mid infrared spectroscopy
Biochemistry, 2003Co-Authors: Marie Louise Groot, Michael A. Van Der Horst, Ivo H M Van Stokkum, Delmar S Larsen, Klaas J. Hellingwerf, Luuk J G W Van Wilderen, Rienk Van GrondelleAbstract:Photoactive yellow protein (PYP) is a bacterial blue light sensor that induces Halorhodospira Halophila to swim away from intense blue light. Light absorption by PYP's intrinsic chromophore, p-coumaric acid, leads to the initiation of a photocycle that comprises several distinct intermediates. Here we describe the initial structural changes of the chromophore and its nearby amino acids, using visible pump/mid-infrared probe spectroscopy. Upon photoexcitation, the trans bands of the chromophore are bleached, and shifts of the phenol ring bands occur. The latter are ascribed to charge translocation, which probably plays an essential role in driving the trans to cis isomerization process. We conclude that breaking of the hydrogen bond of the chromophore's CO group with amino acid Cys69 and formation of a stable cis ground state occur in ∼2 ps. Dynamic changes also include rearrangements of the hydrogen-bonding network of the amino acids around the chromophore. Relaxation of the coumaryl tail of the chromopho...
Marie Louise Groot - One of the best experts on this subject based on the ideXlab platform.
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ultrafast infrared spectroscopy reveals a key step for successful entry into the photocycle for photoactive yellow protein
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: L J G W Van Wilderen, Ivo H M Van Stokkum, Klaas J. Hellingwerf, M. A. Van Der Horst, R. Van Grondelle, Marie Louise GrootAbstract:Photoactive proteins such as PYP (photoactive yellow protein) are generally accepted as model systems for studying protein signal state formation. PYP is a blue-light sensor from the bacterium Halorhodospira Halophila . The formation of PYP9s signaling state is initiated by trans-cis isomerization of the p -coumaric acid chromophore upon the absorption of light. The quantum yield of signaling state formation is ≈0.3. Using femtosecond visible pump/mid-IR probe spectroscopy, we investigated the structure of the very short-lived ground state intermediate (GSI) that results from an unsuccessful attempt to enter the photocycle. This intermediate and the first stable GSI on pathway into the photocycle, I 0 , both have a mid-IR difference spectrum that is characteristic of a cis isomer, but only the I 0 intermediate has a chromophore with a broken hydrogen bond with the backbone N atom of Cys-69. We suggest, therefore, that breaking this hydrogen bond is decisive for a successful entry into the photocycle. The chromophore also engages in a hydrogen-bonding network by means of its phenolate group with residues Tyr-42 and Glu-46. We have investigated the role of this hydrogen bond by exchanging the H bond-donating residue Glu-46 with the weaker H bond-donating glutamine (i.e., Gln-46). We have observed that this mutant exhibits virtually identical kinetics and product yields as WT PYP, even though during the I 0 -to-I 1 transition, on the 800-ps time scale, the hydrogen bond of the chromophore with Gln-46 is broken, whereas this hydrogen bond remains intact with Glu-46.
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initial steps of signal generation in photoactive yellow protein revealed with femtosecond mid infrared spectroscopy
Biochemistry, 2003Co-Authors: Marie Louise Groot, Michael A. Van Der Horst, Ivo H M Van Stokkum, Delmar S Larsen, Klaas J. Hellingwerf, Luuk J G W Van Wilderen, Rienk Van GrondelleAbstract:Photoactive yellow protein (PYP) is a bacterial blue light sensor that induces Halorhodospira Halophila to swim away from intense blue light. Light absorption by PYP's intrinsic chromophore, p-coumaric acid, leads to the initiation of a photocycle that comprises several distinct intermediates. Here we describe the initial structural changes of the chromophore and its nearby amino acids, using visible pump/mid-infrared probe spectroscopy. Upon photoexcitation, the trans bands of the chromophore are bleached, and shifts of the phenol ring bands occur. The latter are ascribed to charge translocation, which probably plays an essential role in driving the trans to cis isomerization process. We conclude that breaking of the hydrogen bond of the chromophore's CO group with amino acid Cys69 and formation of a stable cis ground state occur in ∼2 ps. Dynamic changes also include rearrangements of the hydrogen-bonding network of the amino acids around the chromophore. Relaxation of the coumaryl tail of the chromopho...
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Accelerated Publications Initial Steps of Signal Generation in Photoactive Yellow Protein Revealed with
2003Co-Authors: Femtosecond Mid-infrared Spectroscopy, Marie Louise Groot, Delmar S Larsen, Michael A. Van Der HorstAbstract:ABSTRACT: Photoactive yellow protein (PYP) is a bacterial blue light sensor that induces Halorhodospira Halophila to swim away from intense blue light. Light absorption by PYP’s intrinsic chromophore, p-coumaric acid, leads to the initiation of a photocycle that comprises several distinct intermediates. Here we describe the initial structural changes of the chromophore and its nearby amino acids, using visible pump/mid-infrared probe spectroscopy. Upon photoexcitation, the trans bands of the chromophore are bleached, and shifts of the phenol ring bands occur. The latter are ascribed to charge translocation, which probably plays an essential role in driving the trans to cis isomerization process. We conclude that breaking of the hydrogen bond of the chromophore’s CdO group with amino acid Cys69 and formation of a stable cis ground state occur in 2 ps. Dynamic changes also include rearrangements of the hydrogen-bonding network of the amino acids around the chromophore. Relaxation of the coumaryl tail of the chromophore occurs in 0.9-1 ns, which event we identify with the I0 to I1 transition observed in visible spectroscopy. Activation of PYP1 by blue light induces a negative phototactic response that enables the bacterium Halo-rhodospira Halophila to swim away from harmful exposure to intense blue light (see, for a review, ref 1). Due to its ric
Rienk Van Grondelle - One of the best experts on this subject based on the ideXlab platform.
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Initial photoinduced dynamics of the photoactive yellow protein.
Chemphyschem : a European journal of chemical physics and physical chemistry, 2005Co-Authors: Delmar S Larsen, Rienk Van GrondelleAbstract:The photoactive yellow protein (PYP) is the photoreceptor protein responsible for initiating the blue-light repellent response of the Halorhodospira Halophila bacterium. Optical excitation of the intrinsic chromophore in PYP, p-coumaric acid, leads to the initiation of a photocycle that comprises several distinct intermediates. The dynamical processes responsible for the initiation of the PYP photocycle have been explored with several time-resolved techniques, which include ultrafast electronic and vibrational spectroscopies Ultrafast electronic spectroscopies, such as pump-visible probe, pump-dump-visible probe, and fluorescence upconversion, are useful in identifying the timescales and connectivity of the transient intermediates, while ultrafast vibrational spectroscopies link these intermediates to dynamic structures therein, we present the use of these techniques for exploring the initial dynamics of PYP and show how these techniques provide the basis for understanding the complex relationship between protein and chromophore, which ultimately results in biological function. © 2005 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim.
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Incoherent manipulation of the photoactive yellow protein photocycle with dispersed pump-dump-probe spectroscopy
Biophysical Journal, 2004Co-Authors: Delmar S Larsen, Michael A. Van Der Horst, Ivo H M Van Stokkum, Frank L De Weerd, Mikas Vengris, Klaas J. Hellingwerf, Rienk Van GrondelleAbstract:Photoactive yellow protein is the protein responsible for initiating the "blue-light vision" of Halorhodospira Halophila. The dynamical processes responsible for triggering the photoactive yellow protein photocycle have been disentangled with the use of a novel application of dispersed ultrafast pump-dump-probe spectroscopy, where the photocycle can be started and interrupted with appropriately tuned and timed laser pulses. This "incoherent" manipulation of the photocycle allows for the detailed spectroscopic investigation of the underlying photocycle dynamics and the construction of a fully self-consistent dynamical model. This model requires three kinetically distinct excited-state intermediates, two (ground-state) photocycle intermediates, I0and pR, and a ground-state intermediate through which the protein, after unsuccessful attempts at initiating the photocycle, returns to the equilibrium ground state. Also observed is a previously unknown two-photon ionization channel that generates a radical and an ejected electron into the protein environment. This second excitation pathway evolves simultaneously with the pathway containing the one-photon photocycle intermediates.
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initial steps of signal generation in photoactive yellow protein revealed with femtosecond mid infrared spectroscopy
Biochemistry, 2003Co-Authors: Marie Louise Groot, Michael A. Van Der Horst, Ivo H M Van Stokkum, Delmar S Larsen, Klaas J. Hellingwerf, Luuk J G W Van Wilderen, Rienk Van GrondelleAbstract:Photoactive yellow protein (PYP) is a bacterial blue light sensor that induces Halorhodospira Halophila to swim away from intense blue light. Light absorption by PYP's intrinsic chromophore, p-coumaric acid, leads to the initiation of a photocycle that comprises several distinct intermediates. Here we describe the initial structural changes of the chromophore and its nearby amino acids, using visible pump/mid-infrared probe spectroscopy. Upon photoexcitation, the trans bands of the chromophore are bleached, and shifts of the phenol ring bands occur. The latter are ascribed to charge translocation, which probably plays an essential role in driving the trans to cis isomerization process. We conclude that breaking of the hydrogen bond of the chromophore's CO group with amino acid Cys69 and formation of a stable cis ground state occur in ∼2 ps. Dynamic changes also include rearrangements of the hydrogen-bonding network of the amino acids around the chromophore. Relaxation of the coumaryl tail of the chromopho...
Delmar S Larsen - One of the best experts on this subject based on the ideXlab platform.
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Bifurcation in the Ultrafast Dynamics of the Photoactive Yellow Proteins from Leptospira biflexa and Halorhodospira Halophila
2016Co-Authors: Tyler L Mix, Mikas Vengris, Julia S Kirpich, Masato Kumauchi, Jie Ren, Wouter D. Hoff, Delmar S LarsenAbstract: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
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Noncanonical Photocycle Initiation Dynamics of the Photoactive Yellow Protein (PYP) Domain of the PYP-Phytochrome-Related (Ppr) Photoreceptor
2016Co-Authors: Tyler L Mix, Wouter D Hoff, Masato Kumauchi, Miwa Hara, Rachana Rathod, Delmar S LarsenAbstract: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
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Subpicosecond Excited-State Proton Transfer Preceding Isomerization During the Photorecovery of Photoactive Yellow Protein
2015Co-Authors: Elizabeth C Carroll, Ivo H M Van Stokkum, Wouter D Hoff, Masato Kumauchi, Sang-hun Song, Askat Jailaubekov, Delmar S LarsenAbstract:The ultrafast excited-state dynamics underlying the receptor state photorecovery is resolved in the M100A mutant of the photoactive yellow protein (PYP) from Halorhodospira Halophila. The M100A PYP mutant, with its distinctly slower photocycle than wt PYP, allows isolation of the pB signaling state for study of the photodynamics of the protonated chromophore cis-p-coumaric acid. Transient absorption signals indicate a subpicosecond excited-state proton-transfer reaction in the pB state that results in chromophore deprotonation prior to the cis−trans isomerization required in the photorecovery dynamics of the pG state. Two terminal photoproducts are observed, a blue-absorbing species presumed to be deprotonated trans-p-coumaric acid and an ultraviolet-absorbing protonated photoproduct. These two photoproducts are hypothesized to originate from an equilibrium of open and closed folded forms of the signaling state, I2 and I2′
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Initial photoinduced dynamics of the photoactive yellow protein.
Chemphyschem : a European journal of chemical physics and physical chemistry, 2005Co-Authors: Delmar S Larsen, Rienk Van GrondelleAbstract:The photoactive yellow protein (PYP) is the photoreceptor protein responsible for initiating the blue-light repellent response of the Halorhodospira Halophila bacterium. Optical excitation of the intrinsic chromophore in PYP, p-coumaric acid, leads to the initiation of a photocycle that comprises several distinct intermediates. The dynamical processes responsible for the initiation of the PYP photocycle have been explored with several time-resolved techniques, which include ultrafast electronic and vibrational spectroscopies Ultrafast electronic spectroscopies, such as pump-visible probe, pump-dump-visible probe, and fluorescence upconversion, are useful in identifying the timescales and connectivity of the transient intermediates, while ultrafast vibrational spectroscopies link these intermediates to dynamic structures therein, we present the use of these techniques for exploring the initial dynamics of PYP and show how these techniques provide the basis for understanding the complex relationship between protein and chromophore, which ultimately results in biological function. © 2005 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim.
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Incoherent manipulation of the photoactive yellow protein photocycle with dispersed pump-dump-probe spectroscopy
Biophysical Journal, 2004Co-Authors: Delmar S Larsen, Michael A. Van Der Horst, Ivo H M Van Stokkum, Frank L De Weerd, Mikas Vengris, Klaas J. Hellingwerf, Rienk Van GrondelleAbstract:Photoactive yellow protein is the protein responsible for initiating the "blue-light vision" of Halorhodospira Halophila. The dynamical processes responsible for triggering the photoactive yellow protein photocycle have been disentangled with the use of a novel application of dispersed ultrafast pump-dump-probe spectroscopy, where the photocycle can be started and interrupted with appropriately tuned and timed laser pulses. This "incoherent" manipulation of the photocycle allows for the detailed spectroscopic investigation of the underlying photocycle dynamics and the construction of a fully self-consistent dynamical model. This model requires three kinetically distinct excited-state intermediates, two (ground-state) photocycle intermediates, I0and pR, and a ground-state intermediate through which the protein, after unsuccessful attempts at initiating the photocycle, returns to the equilibrium ground state. Also observed is a previously unknown two-photon ionization channel that generates a radical and an ejected electron into the protein environment. This second excitation pathway evolves simultaneously with the pathway containing the one-photon photocycle intermediates.
