Ultrafast mid-infrared spectroscopy by chirped pulse upconversion in 1800-1000cm(-1) region

Broadband femtosecond mid-infrared pulses can be converted into the visible spectral region by chirped pulse upconversion. We report here the upconversion of pump probe transient signals in the frequency region below 1800c

Molecular Adaptation of Photoprotection: Triplet States in Light-Harvesting Proteins

The photosynthetic light-harvesting systems of purple bacteria and plants both utilize specific carotenoids as quenchers of the harmful (bacterio)chlorophyll triplet states via triplet-triplet energy transfer. Here, we explore how the binding of carotenoids to the different types of light-harvesting proteins found in plants and purple bacteria provides adaptation in this vital photoprotective function. We show that the creation of the carotenoid triplet states in the light-harvesting complexes may occur without detectable conformational changes, in contrast to that found for carotenoids in solution. However, in plant light-harvesting complexes, the triplet wavefunction is shared between the carotenoids and their adjacent chlorophylls. This is not observed for the antenna proteins of purple bacteria, where the triplet is virtually fully located on the carotenoid molecule. These results explain the faster triplet-triplet transfer times in plant light-harvesting complexes. We show that this molecular mechanism, which spreads the location of the triplet wavefunction through the pigments of plant light-harvesting complexes, results in the absence of any detectable chlorophyll triplet in these complexes upon excitation, and we propose that it emerged as a photoprotective adaptation during the evolution of oxygenic photosynthesis

Photoinduced transformation of UVR8 monitored by vibrational and fluorescence spectroscopy

Tryptophan residues at the dimer interface of the plant photoreceptor UVR8 promote monomerisation after UV-B absorption via a so far unknown mechanism. Using FTIR spectroscopy we assign light-induced structural transitions of UVR8 mainly to amino acid side chains without major transformations of the secondary structure of the physiologically relevant C-terminal extension. Additionally, we assign the monomerisation associated increase and red shift of the UVR8 tryptophan emission to a photoinduced rearrangement of tryptophan side chains and a relocation of the aspartic acid residues D96 and D107, respectively. By illumination dependent emission spectroscopy we furthermore determined the quantum yield of photoinduced monomerisation to 20 ± 8%

The molecular pH-response mechanism of the plant light-stress sensor PsbS

Plants need to protect themselves from excess light, which causes photo-oxidative damage and lowers the efficiency of photosynthesis. Photosystem II subunit S (PsbS) is a pH sensor protein that plays a crucial role in plant photoprotection by detecting thylakoid lumen acidification in excess light conditions via two lumen-faced glutamates. However, how PsbS is activated under low-pH conditions is unknown. To reveal the molecular response of PsbS to low pH, here we perform an NMR, FTIR and 2DIR spectroscopic analysis of Physcomitrella patens PsbS and of the E176Q mutant in which an active glutamate has been replaced. The PsbS response mechanism at low pH involves the concerted action of repositioning of a short amphipathic helix containing E176 facing the lumen and folding of the luminal loop fragment adjacent to E71 to a 310-helix, providing clear evidence of a conformational pH switch. We propose that this concerted mechanism is a shared motif of proteins of the light-harvesting family that may control thylakoid inter-protein interactions driving photoregulatory responses

Helical Contributions Mediate Light-Activated Conformational Change in the LOV2 Domain of Avena sativa Phototropin 1

Algae, plants, bacteria, and fungi contain flavin-binding light-oxygen-voltage (LOV) domains that function as blue light sensors to control cellular responses to light. In the second LOV domain of phototropins, called LOV2 domains, blue light illumination leads to covalent bond formation between protein and flavin that induces the dissociation and unfolding of a C-terminally attached α helix (Jα) and the N-terminal helix (A′α). To date, the majority of studies on these domains have focused on versions that contain truncations in the termini, which creates difficulties when extrapolating to the much larger proteins that contain these domains. Here, we study the influence of deletions and extensions of the A′α helix of the LOV2 domain of Avena sativa phototropin 1 (AsLOV2) on the light-triggered structural response of the protein by Fourier-transform infrared difference spectroscopy. Deletion of the A′α helix abolishes the light-induced unfolding of Jα, whereas extensions of the A′α helix lead to an attenuated structural change of Jα. These results are different from shorter constructs, indicating that the conformational changes in full-length phototropin LOV domains might not be as large as previously assumed, and that the well-characterized full unfolding of the Jα helix in AsLOV2 with short A′α helices may be considered a truncation artifact. It also suggests that the N- and C-terminal helices of phot-LOV2 domains are necessary for allosteric regulation of the phototropin kinase domain and may provide a basis for signal integration of LOV1 and LOV2 domains in phototropins

Lower frequency region mid-infrared spectroscopy by chirped pulse upconversion

UV/visible pump, mid-IR probe spectroscopy measurements based on the chirped upconversion method were expanded to the frequency region below 1800cm−1 with the nonlinear optical crystal AgGaGeS4. Pump-probe experiments were demonstrated with GaAs and the photoreceptor protein Slr1694

Two-photon Absorption and Photoionization of a Bacterial Phytochrome

Phytochromes constitute a family of photosensory proteins that are utilized by various organisms to regulate several physiological processes. Phytochromes bind a bilin pigment that switches its isomeric state upon absorption of red or far-red photons, resulting in protein conformational changes that are sensed by the organism. Previously, the ultrafast dynamics in bacterial phytochrome was resolved to atomic resolution by time-resolved serial femtosecond X-ray diffraction (TR-SFX), showing extensive changes in its molecular conformation at 1 picosecond delay time. However, the large excitation fluence of mJ/mm2 used in TR-SFX questions the validity of the observed dynamics. In this work, we present an excitation-dependent ultrafast transient absorption study to test the response of a related bacterial phytochrome to excitation fluence. We observe excitation power-dependent sub-picosecond dynamics, assigned to the population of high-lying excited state Sn through resonantly enhanced two-photon absorption, followed by rapid internal conversion to the low-lying S1 state. Inspection of the long-lived spectrum under high fluence shows that in addition to the primary intermediate Lumi-R, spectroscopic signatures of solvated electrons and ionized chromophore radicals are observed. Supported by numerical modelling, we propose that under excitation fluences of tens of μJ/mm2 and higher, bacterial phytochrome partly undergoes photoionization from the Sn state in competition with internal conversion to the S1 state in 300 fs. We suggest that the extensive structural changes of related, shorter bacterial phytochrome, lacking the PHY domain, resolved from TR-SFX may have been affected by the ionized species. We propose approaches to minimize the two-photon absorption process by tuning the excitation spectrum away from the S1 absorption or using phytochromes exhibiting minimized or shifted S1 absorption.</p

Molecular Origin of Photoprotection in Cyanobacteria Probed by Watermarked Femtosecond Stimulated Raman Spectroscopy

Photoprotection is fundamental in photosynthesis to avoid oxidative photodamage upon excess light exposure. Excited chlorophylls (Chl) are quenched by carotenoids, but the precise molecular origin remains controversial. The cyanobacterial HliC protein belongs to the Hlip family ancestral to plant light-harvesting complexes, and binds Chl a and β-carotene in 2:1 ratio. We analyzed HliC by watermarked femtosecond stimulated Raman spectroscopy to follow the time evolution of its vibrational modes. We observed a 2 ps rise of the C=C stretch band of the 2Ag - (S1) state of β-carotene upon Chl a excitation, demonstrating energy transfer quenching and fast excess-energy dissipation. We detected two distinct β-carotene conformers by the C=C stretch frequency of the 2Ag - (S1) state, but only the β-carotene whose 2Ag - energy level is significantly lowered and has a lower C=C stretch frequency is involved in quenching. It implies that the low carotenoid S1 energy that results from specific pigment-protein or pigment-pigment interactions is the key property for creating a dissipative energy channel. We conclude that watermarked femtosecond stimulated Raman spectroscopy constitutes a promising experimental method to assess energy transfer and quenching mechanisms in oxygenic photosynthesis

Strong pH-Dependent Near-Infrared Fluorescence in a Microbial Rhodopsin Reconstituted with a Red-Shifting Retinal Analogue

Contains fulltext : 200410.pdf (Publisher’s version ) (Open Access