41 research outputs found
Salt shock-inducible Photosystem I cyclic electron transfer in Synechocystis PCC6803 relies on binding of ferredoxin:NADP+ reductase to the thylakoid membranes via its CpcD phycobilisome-linker homologous N-terminal domain
AbstractRelative to ferredoxin:NADP+ reductase (FNR) from chloroplasts, the comparable enzyme in cyanobacteria contains an additional 9 kDa domain at its amino-terminus. The domain is homologous to the phycocyanin associated linker polypeptide CpcD of the light harvesting phycobilisome antennae. The phenotypic consequences of the genetic removal of this domain from the petH gene, which encodes FNR, have been studied in Synechocystis PCC 6803. The in frame deletion of 75 residues at the amino-terminus, rendered chloroplast length FNR enzyme with normal functionality in linear photosynthetic electron transfer. Salt shock correlated with increased abundance of petH mRNA in the wild-type and mutant alike. The truncation stopped salt stress-inducible increase of Photosystem I-dependent cyclic electron flow. Both photoacoustic determination of the storage of energy from Photosystem I specific far-red light, and the re-reduction kinetics of P700+, suggest lack of function of the truncated FNR in the plastoquinoneâcytochrome b6f complex reductase step of the PS I-dependent cyclic electron transfer chain. Independent gold-immunodecoration studies and analysis of FNR distribution through activity staining after native polyacrylamide gelelectrophoresis showed that association of FNR with the thylakoid membranes of Synechocystis PCC 6803 requires the presence of the extended amino-terminal domain of the enzyme. The truncated ÎpetH gene was also transformed into a NAD(P)H dehydrogenase (NDH1) deficient mutant of Synechocystis PCC 6803 (strain M55) (T. Ogawa, Proc. Natl. Acad. Sci. USA 88 (1991) 4275â4279). Phenotypic characterisation of the double mutant supported our conclusion that both the NAD(P)H dehydrogenase complex and FNR contribute independently to the quinone cytochrome b6f reductase step in PS I-dependent cyclic electron transfer. The distribution, binding properties and function of FNR in the model cyanobacterium Synechocystis PCC 6803 will be discussed
The Short-Lived Signaling State of the Photoactive Yellow Protein Photoreceptor Revealed by Combined Structural Probes
The signaling state of the photoactive yellow protein (PYP) photoreceptor is transiently developed via isomerization of its blue-light-absorbing chromophore. The associated structural rearrangements have large amplitude but, due to its transient nature and chemical exchange reactions that complicate NMR detection, its accurate three-dimensional structure in solution has been elusive. Here we report on direct structural observation of the transient signaling state by combining double electron electron resonance spectroscopy (DEER), NMR, and time-resolved pump-probe X-ray solution scattering (TR-SAXS/WAXS). Measurement of distance distributions for doubly spin-labeled photoreceptor constructs using DEER spectroscopy suggests that the signaling state is well ordered and shows that interspin-label distances change reversibly up to 19 Ă
upon illumination. The SAXS/WAXS difference signal for the signaling state relative to the ground state indicates the transient formation of an ordered and rearranged conformation, which has an increased radius of gyration, an increased maximum dimension, and a reduced excluded volume. Dynamical annealing calculations using the DEER derived long-range distance restraints in combination with short-range distance information from (1)H-(15)N HSQC perturbation spectroscopy give strong indication for a rearrangement that places part of the N-terminal domain in contact with the exposed chromophore binding cleft while the terminal residues extend away from the core. Time-resolved global structural information from pump-probe TR-SAXS/WAXS data supports this conformation and allows subsequent structural refinement that includes the combined energy terms from DEER, NMR, and SAXS/WAXS together. The resulting ensemble simultaneously satisfies all restraints, and the inclusion of TR-SAXS/WAXS effectively reduces the uncertainty arising from the possible spin-label orientations. The observations are essentially compatible with reduced folding of the I(2)' state (also referred to as the 'pB' state) that is widely reported, but indicates it to be relatively ordered and rearranged. Furthermore, there is direct evidence for the repositioning of the N-terminal region in the I(2)' state, which is structurally modeled by dynamical annealing and refinement calculations
Molecular Activation Mechanism and Structural Dynamics of Orange Carotenoid Protein
Like most photosynthetic organisms, cyanobacteria are vulnerable to fluctuations in light intensity, which can damage their photosynthetic machinery. To protect against this, they use a photoprotective mechanism called non-photochemical quenching (NPQ), where excess absorbed photo-energy is dissipated as heat. In cyanobacteria, light activation of Orange Carotenoid Protein (OCP) is the critical first step in the NPQ response. OCP is also the only known photosensitive protein, which uses carotenoid for its activation. We summarize the current knowledge on the light induced reactions of OCP; the different mechanisms of activation that have been proposed; photocycle kinetics and characteristics; and the reported structural intermediates. We discuss the possible interpretations of reported experimental results, and we formulate important open questions and directions for future work, to reveal the molecular and structural basis of photosensing by OCP
Molecular Activation Mechanism and Structural Dynamics of Orange Carotenoid Protein
Like most photosynthetic organisms, cyanobacteria are vulnerable to fluctuations in light intensity, which can damage their photosynthetic machinery. To protect against this, they use a photoprotective mechanism called non-photochemical quenching (NPQ), where excess absorbed photo-energy is dissipated as heat. In cyanobacteria, light activation of Orange Carotenoid Protein (OCP) is the critical first step in the NPQ response. OCP is also the only known photosensitive protein, which uses carotenoid for its activation. We summarize the current knowledge on the light induced reactions of OCP; the different mechanisms of activation that have been proposed; photocycle kinetics and characteristics; and the reported structural intermediates. We discuss the possible interpretations of reported experimental results, and we formulate important open questions and directions for future work, to reveal the molecular and structural basis of photosensing by OCP
Linear and Non-Linear Population Retrieval with Femtosecond Optical Pumping of Molecular Crystals for the Generalised Uniaxial and Biaxial Systems
Femtosecond optical measurements of photoexcitable molecular crystals carry ultrafast dynamics information with structural sensitivity. The creation and detection of transient dynamics depend on the optical parameters, as well as the explicit molecular structure, crystal symmetry, crystal orientation, polarisation of the photoexciting beam, and interaction geometry. In order to retrieve the linear and non-linear population transfer in photoexcited crystals, excitation theory is combined here with the calculation of birefringence decomposition and is shown for both the generalised uniaxial and biaxial systems. A computational tool was constructed based on this treatment to allow modelling of electric field decomposition, dipole projections, and non-linear excitation population levels. This is available open source and with a GUI for ease of use. Such work has applications in two areas of ultrafast science: multidimensional optical crystallography and femtosecond time-resolved X-ray crystallography
Photoisomerization and Proton Transfer in the Forward and Reverse Photoswitching of the Fast-Switching M159T Mutant of the Dronpa Fluorescent Protein
The
fast-switching M159T mutant of the reversibly photoswitchable
fluorescent protein Dronpa has an enhanced yield for the on-to-off
reaction. The forward and reverse photoreactions proceed via cisâtrans
and transâcis photoisomerization, yet protonation and deprotonation
of the hydroxyphenyl oxygen of the chromophore is responsible for
the majority of the resulting spectroscopic contrast. Ultrafast visible-pump,
infrared-probe spectroscopy was used to detect the picosecond, nanosecond,
as well as metastable millisecond intermediates. Additionally, static
FTIR difference measurements of the Dronpa-M159T mutant correspond
very closely to those of the wild type Dronpa, identifying the p-hydroxybenzylidene-imidazolinone
chromophore in the cis anion and trans neutral forms in the bright
âonâ and dark âoffâ states, respectively.
Green excitation of the on state is followed by dominant radiative
decay with characteristic time constants of 1.9 ps, 185 ps, and 1.1
ns, and additionally reveals spectral changes belonging to the species
decaying with a 1.1 ns time constant, associated with both protein
and chromophore modes. A 1 ms measurement of the on state identifies
bleach features that correspond to those seen in the static off-minus-on
Fourier transform infrared (FTIR) difference spectrum, indicating
that thermal protonation of the hydroxyphenyl oxygen proceeds within
this time window. Blue excitation of the off state directly resolves
the formation of the primary photoproduct with 0.6 and 14 ps time
constants, which is stable on the nanosecond time scale. Assignment
of the primary photoproduct to the cis neutral chromophore in the
electronic ground state is supported by the frequency positions expected
relative to those for the nonplanar distorted geometry for the off
state. A 1 ms measurement of the off state corresponds closely with
the on-minus-off FTIR difference spectrum, indicating thermal deprotonation
and rearrangement of the Arg66 side chain to be complete
Open hardware microsecond dispersive transient absorption spectrometer for linear optical response
Photoisomerization and Proton Transfer in the Forward and Reverse Photoswitching of the Fast-Switching M159T Mutant of the Dronpa Fluorescent Protein
The
fast-switching M159T mutant of the reversibly photoswitchable
fluorescent protein Dronpa has an enhanced yield for the on-to-off
reaction. The forward and reverse photoreactions proceed via cisâtrans
and transâcis photoisomerization, yet protonation and deprotonation
of the hydroxyphenyl oxygen of the chromophore is responsible for
the majority of the resulting spectroscopic contrast. Ultrafast visible-pump,
infrared-probe spectroscopy was used to detect the picosecond, nanosecond,
as well as metastable millisecond intermediates. Additionally, static
FTIR difference measurements of the Dronpa-M159T mutant correspond
very closely to those of the wild type Dronpa, identifying the p-hydroxybenzylidene-imidazolinone
chromophore in the cis anion and trans neutral forms in the bright
âonâ and dark âoffâ states, respectively.
Green excitation of the on state is followed by dominant radiative
decay with characteristic time constants of 1.9 ps, 185 ps, and 1.1
ns, and additionally reveals spectral changes belonging to the species
decaying with a 1.1 ns time constant, associated with both protein
and chromophore modes. A 1 ms measurement of the on state identifies
bleach features that correspond to those seen in the static off-minus-on
Fourier transform infrared (FTIR) difference spectrum, indicating
that thermal protonation of the hydroxyphenyl oxygen proceeds within
this time window. Blue excitation of the off state directly resolves
the formation of the primary photoproduct with 0.6 and 14 ps time
constants, which is stable on the nanosecond time scale. Assignment
of the primary photoproduct to the cis neutral chromophore in the
electronic ground state is supported by the frequency positions expected
relative to those for the nonplanar distorted geometry for the off
state. A 1 ms measurement of the off state corresponds closely with
the on-minus-off FTIR difference spectrum, indicating thermal deprotonation
and rearrangement of the Arg66 side chain to be complete
Singular values.
<p>The singular values for the heterogeneous decay and dynamic motion data sets in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1005528#pcbi.1005528.g001" target="_blank">Fig 1B and 1D</a>. Note that the heterogeneous decay data has three significant values and the dynamic motion data has two.</p