scholarly journals Time-resolved fluorescence analysis of LHCII in the presence of PsbS at neutral and low pH

2018 ◽  
Author(s):  
Emanuela Crisafi ◽  
Maithili Krishnan ◽  
Anjali Pandit
Keyword(s):  
Lipid Membranes ◽  
Light Stress ◽  
Fluorescence Analysis ◽  
Low Ph ◽  
Photochemical Quenching ◽  
Ph Sensing ◽  
Fluorescence Lifetimes ◽  
Light Harvesting Complexes ◽  
Time Resolved

In plant chloroplast membranes, non-photochemical quenching (NPQ) is activated as a response to a low luminal pH and controlled by the pH-sensing protein PsbS. It has been proposed that PsbS directly interacts with the light-harvesting complexes (LHCII) of Photosystem II, inducing quenching of LHCII Chl excitations, whilst others proposed that PsbS has an indirect role in controlling the organization of the membrane. In this study, we systematically test the influence of low pH and PsbS on the fluorescence lifetimes of membrane-embedded spinach LHCII. The proteoliposome preparations contain LHCII in mild quenched states, aimed to mimic fluorescence conditions of dark-adapted leaves. We find that under those conditions, acidification and the presence of PsbS do not have significant effect on the LHCII Chl fluorescence lifetimes. This supports a view in which the functional role of PsbS consists of re-organizing the thylakoid membrane under light stress, rather than creating direct quencher states. The dimeric form of PsbS appears to be destabilized in lipid membranes compared to detergent micelles, which might explain why the low-pH PsbS crystal structure is dimeric, while in vivo activation of PsbS has been correlated with its monomerization at low pH.

scholarly journals Identification of parallel pH- and zeaxanthin-dependent quenching of excess energy in LHCSR3 in Chlamydomonas reinhardtii

2020 ◽  
Author(s):  
Julianne M. Troiano ◽  
Federico Perozeni ◽  
Raymundo Moya ◽  
Luca Zuliani ◽  
Kwangryul Baek ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Light Harvesting ◽  
Complex Stress ◽  
Excess Energy ◽  
Photochemical Quenching ◽  
Light Harvesting Complexes ◽  
Light Harvesting Complex ◽  
Non Photochemical Quenching

AbstractUnder high light conditions, oxygenic photosynthetic organisms avoid photodamage by thermally dissipating excess absorbed energy, which is called non-photochemical quenching (NPQ). In green algae, a chlorophyll and carotenoid-binding protein, light-harvesting complex stress-related (LHCSR3), detects excess energy via pH and serves as a quenching site. However, the mechanisms by which LHCSR3 functions have not been determined. Using a combined in vivo and in vitro approach, we identify two parallel yet distinct quenching processes, individually controlled by pH and carotenoid composition, and their likely molecular origin within LHCSR3 from Chlamydomonas reinhardtii. The pH-controlled quenching is removed within a mutant LHCSR3 that lacks the protonable residues responsible for sensing pH. Constitutive quenching in zeaxanthin-enriched systems demonstrates zeaxanthin-controlled quenching, which may be shared with other light-harvesting complexes. We show that both quenching processes prevent the formation of damaging reactive oxygen species, and thus provide distinct timescales and mechanisms of protection in a changing environment.

scholarly journals From isolated light-harvesting complexes to the thylakoid membrane: a single-molecule perspective

Nanophotonics ◽  
2018 ◽  
Vol 7 (1) ◽  
pp. 81-92 ◽  
Author(s):  
J. Michael Gruber ◽  
Pavel Malý ◽  
Tjaart P.J. Krüger ◽  
Rienk van Grondelle
Keyword(s):  
Single Molecule ◽  
Thylakoid Membrane ◽  
Light Harvesting ◽  
Protein Complexes ◽  
Conformational Dynamics ◽  
Physical Models ◽  
Photochemical Quenching ◽  
Light Harvesting Complexes ◽  
Fluorescence Measurements

AbstractThe conversion of solar radiation to chemical energy in plants and green algae takes place in the thylakoid membrane. This amphiphilic environment hosts a complex arrangement of light-harvesting pigment-protein complexes that absorb light and transfer the excitation energy to photochemically active reaction centers. This efficient light-harvesting capacity is moreover tightly regulated by a photoprotective mechanism called non-photochemical quenching to avoid the stress-induced destruction of the catalytic reaction center. In this review we provide an overview of single-molecule fluorescence measurements on plant light-harvesting complexes (LHCs) of varying sizes with the aim of bridging the gap between the smallest isolated complexes, which have been well-characterized, and the native photosystem. The smallest complexes contain only a small number (10–20) of interacting chlorophylls, while the native photosystem contains dozens of protein subunits and many hundreds of connected pigments. We discuss the functional significance of conformational dynamics, the lipid environment, and the structural arrangement of this fascinating nano-machinery. The described experimental results can be utilized to build mathematical-physical models in a bottom-up approach, which can then be tested on larger in vivo systems. The results also clearly showcase the general property of biological systems to utilize the same system properties for different purposes. In this case it is the regulated conformational flexibility that allows LHCs to switch between efficient light-harvesting and a photoprotective function.

Time-Resolved Activation of pH Sensing and Imaging in Vivo by a Remotely Controllable DNA Nanomachine

Nano Letters ◽  
2019 ◽  
Vol 20 (2) ◽  
pp. 874-880 ◽  
Author(s):  
Jian Zhao ◽  
Yinghao Li ◽  
Mingming Yu ◽  
Zhanjun Gu ◽  
Lele Li ◽  
...  
Keyword(s):  
Ph Sensing ◽  
Time Resolved ◽  
Dna Nanomachine

scholarly journals In Vivo Resolution of Multiexponential Decays of Multiple Near-Infrared Molecular Probes by Fluorescence Lifetime-Gated Whole-Body Time-Resolved Diffuse Optical Imaging

2007 ◽  
Vol 6 (4) ◽  
pp. 7290.2007.00020 ◽  
Author(s):  
Walter Akers ◽  
Frederic Lesage ◽  
Dewey Holten ◽  
Samuel Achilefu
Keyword(s):  
Optical Imaging ◽  
Fluorescence Lifetime ◽  
Fluorescence Intensity ◽  
Near Infrared ◽  
Molecular Probes ◽  
Whole Body ◽  
Diffuse Optical Imaging ◽  
Fluorescence Lifetimes ◽  
Time Resolved

The biodistribution of two near-infrared fluorescent agents was assessed in vivo by time-resolved diffuse optical imaging. Bacteriochlorophyll a (BC) and cypate-glysine-arginine-aspartic acid-serine-proline-lysine-OH (Cyp-GRD) were administered separately or combined to mice with subcutaneous xenografts of human breast adenocarcinoma and slow-release estradiol pellets for improved tumor growth. The same excitation (780 nm) and emission (830 nm) wavelengths were used to image the distinct fluorescence lifetime distribution of the fluorescent molecular probes in the mouse cancer model. Fluorescence intensity and lifetime maps were reconstructed after raster-scanning whole-body regions of interest by time-correlated single-photon counting. Each captured temporal point-spread function (TPSF) was deconvolved using both a single and a multiexponental decay model to best determine the measured fluorescence lifetimes. The relative signal from each fluorophore was estimated for any region of interest included in the scanned area. Deconvolution of the individual TPSFs from whole-body fluorescence intensity scans provided corresponding lifetime images for comparing individual component biodistribution. In vivo fluorescence lifetimes were determined to be 0.8 ns (Cyp-GRD) and 2 ns (BC). This study demonstrates that the relative biodistribution of individual fluorophores with similar spectral characteristics can be compartmentalized by using the time-domain fluorescence lifetime gating method.

scholarly journals Time-resolved fluorescence imaging technique for rat brain tumors analysis

2021 ◽  
Vol 2058 (1) ◽  
pp. 012028
Author(s):  
Yu S Maklygina ◽  
I D Romanishkin ◽  
A S Skobeltsin ◽  
T A Savelyeva ◽  
A A Potapov ◽  
...  
Keyword(s):  
Spectroscopic Analysis ◽  
Imaging Technique ◽  
Fluorescence Analysis ◽  
Female Rats ◽  
Time Resolved Fluorescence ◽  
Main Attention ◽  
Time Resolved ◽  
New Approach ◽  
The Difference

Abstract The paper presents a new approach to assessing the state of tissues that differ in phenotype and in the degree of immunocompetent cells activity using photosensitizers (PS) and time-resolved fluorescence analysis methods. The main attention is paid to the detection of differences between tumor cells and tumor-associated macrophages (TAM) using spectroscopic and microscopic methods by the fluorescent kinetics signal and the difference in the accumulation of PS (the accumulation is several times greater in macrophages). The results of the PS photoluminescence study were obtained using two different techniques: time-resolved spectroscopy and time-resolved fluorescence microscopy (FLIM). Time-resolved spectroscopic analysis of the PS fluorescence lifetime was performed on adult female rats with induced C6 glioma in vivo. 5-ALA-induced Pp IX, which is widely used in clinical practice for carrying out effective conduction photodiagnostics and PDT, was used as the PS.

scholarly journals Decay- and evolution-associated spectra of time-resolved fluorescence of LHCII aggregates

2019 ◽  
Vol 58 (4) ◽  
Author(s):  
Andrius Gelzinis ◽  
Yakov Braver ◽  
Jevgenij Chmeliov ◽  
Leonas Valkunas
Keyword(s):  
Global Analysis ◽  
Photosynthetic Apparatus ◽  
State Model ◽  
Multivariate Curve Resolution ◽  
Careful Examination ◽  
Photochemical Quenching ◽  
Time Resolved Fluorescence ◽  
Light Harvesting Complexes ◽  
Time Resolved ◽  
Three State Model

Non-photochemical quenching (NPQ) is responsible for the protection of the photosynthetic apparatus of plants from photodamage at high-light conditions. It is commonly agreed that NPQ takes place in the major light-harvesting complexes (LHCII), however, its exact mechanisms are still under debate. Valuable information about its molecular nature can be provided by measuring time-resolved fluorescence (TRF) spectra of LHCII complexes and their aggregates. Previously [Chmeliov et al., Nat. Plants 2, 16045 (2016)], we analysed the corresponding TRF spectra using the multivariate curve resolution method and proposed a three-state model to describe the spectroscopic data. Usually, such data is described in terms of global analysis resulting in decay or evolution-associated spectra. In this work, we apply such analysis to the TRF data of LHCII aggregates and show that, although mathematically feasible, it cannot be directly related to the physical kinetic model. Nevertheless, a careful examination supplemented with additional spectroscopic information still results in the same three-state model proposed before.

scholarly journals The PsbS protein and low pH are necessary and sufficient to induce quenching in the light-harvesting complex of plants LHCII

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Lauren Nicol ◽  
Roberta Croce
Keyword(s):  
Low Ph ◽  
In Vivo Studies ◽  
Photochemical Quenching ◽  
Photosynthetic Membrane ◽  
Light Harvesting Complex ◽  
In Vivo Experiments ◽  
The One ◽  
Necessary And Sufficient

AbstractPhotosynthesis is tightly regulated in order to withstand dynamic light environments. Under high light intensities, a mechanism known as non-photochemical quenching (NPQ) dissipates excess excitation energy, protecting the photosynthetic machinery from damage. An obstacle that lies in the way of understanding the molecular mechanism of NPQ is the large gap between in vitro and in vivo studies. On the one hand, the complexity of the photosynthetic membrane makes it challenging to obtain molecular information from in vivo experiments. On the other hand, a suitable in vitro system for the study of quenching is not available. Here we have developed a minimal NPQ system using proteoliposomes. With this, we demonstrate that the combination of low pH and PsbS is both necessary and sufficient to induce quenching in LHCII, the main antenna complex of plants. This proteoliposome system can be further exploited to gain more insight into how PsbS and other factors (e.g. zeaxanthin) influence the quenching mechanism observed in LHCII.

Devices and methods for room-temperature fluorescence analysis

1989 ◽  
Vol 323 (1216) ◽  
pp. 241-251 ◽  
Keyword(s):  
Fluorescence Analysis ◽  
Pulse Amplitude ◽  
Photochemical Quenching ◽  
Specific Information ◽  
Pam Fluorometer ◽  
Fluorescence Measurements ◽  
Fluorescence Rise ◽  
Absorbance Changes ◽  
Sun And Shade

An overview of the various types of photochemical and non-photochemical fluorescence quenching in vivo is given. Devices and methods are outlined that allow specific information to be obtained from complex fluorescence responses. The importance of correlated measurements of other, independent photosynthesis signals is emphasized. It is shown that a recently introduced pulse-amplitude modulation fluorometer (PAM fluorometer) can also be used with modified emitter-detector units to measure absorbance changes. Examples are given for absorbance changes of the Hill reagent methyl purple, induced by single turnover flashes, and for P 700 absorbance changes measured simultaneously with fluorescence. Correlated P 700 and fluorescence measurements give deeper insights into the control of electron transfer from PQH 2 to cytochrome (cyt) b / f and into the intersystem acceptor-pool size of sun and shade leaves. Possible explanations for differences in pool sizes determined by P 700 and fluorescence measurements are discussed. By using P 700 reduction as an indicator, it is shown that in saturating light the plastoquinone (PQ) pool is already reduced within 50 ms, whereas the last phase of the fluorescence rise (I 2 -P) takes about 300 ms and is paralleled by the re-reduction of P 700 . It is concluded that I 2 -P reflects removal of photochemical quenching at PSI and that 50 ms saturation pulses are appropriate to eliminate the relevant photochemical PSII quenching.

scholarly journals Aggregation-related quenching of LHCII in liposomes revealed by single-molecule spectroscopy

2020 ◽  
Author(s):  
Marijonas Tutkus ◽  
Jevgenij Chmeliov ◽  
Gediminas Trinkunas ◽  
Parveen Akhtar ◽  
Petar H. Lambrev ◽  
...  
Keyword(s):  
Single Molecule ◽  
Fluorescence Lifetime ◽  
Lipid Membranes ◽  
Photochemical Quenching ◽  
Liposome Size ◽  
Function Relationship ◽  
Structure And Function Relationship ◽  
Protein Density ◽  
And Function

AbstractIncorporation of membrane proteins into reconstituted lipid membranes is a common approach for studying their structure and function relationship in a native-like environment. In this work, we investigated fluorescence properties of liposome-reconstituted LHCII. By utilizing liposome labelling with the fluorescent dye molecules and single-molecule microscopy techniques, we were able to study truly liposome-reconstituted LHCII and compare them with bulk measurements and liposome-free LHCII aggregates on bound surface. Our results showed that fluorescence lifetime in bulk and of that for single liposome measurements were correlated. The fluorescence lifetimes of LHCII were shorter for liposome-free LHCII than for reconstituted LHCII. In the case of liposome-reconstituted LHCII, fluorescence lifetime showed dependence on the protein density reminiscent to concentration quenching. The dependence of fluorescence lifetime of LHCII on the liposome size was not significant. Our results demonstrated that fluorescence quenching can be induced by LHCII-LHCII interactions in reconstituted membranes, most likely occurring via the same mechanism as photoprotective non-photochemical quenching in vivo.

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