Single-molecule spectroscopy of LHCSR1 protein dynamics identifies two distinct states responsible for multi-timescale photosynthetic photoprotection

In oxygenic photosynthesis, light harvesting is regulated to safely dissipate excess energy and prevent the formation of harmful photoproducts. Regulation is known to be necessary for fitness, but the molecular mechanisms are not understood. One challenge has been that ensemble experiments average over active and dissipative behaviours, preventing identification of distinct states. Here, we use single-molecule spectroscopy to uncover the photoprotective states and dynamics of the light-harvesting complex stress-related 1 (LHCSR1) protein, which is responsible for dissipation in green algae and moss. We discover the existence of two dissipative states. We find that one of these states is activated by pH and the other by carotenoid composition, and that distinct protein dynamics regulate these states. Together, these two states enable the organism to respond to two types of intermittency in solar intensity-step changes (clouds and shadows) and ramp changes (sunrise), respectively. Our findings reveal key control mechanisms underlying photoprotective dissipation, with implications for increasing biomass yields and developing robust solar energy devices

Single-molecule identification of quenched and unquenched states of LHCII

In photosynthetic light harvesting, absorbed sunlight is converted to electron flow with near-unity quantum efficiency under low light conditions. Under high light conditions, plants avoid damage to their molecular machinery by activating a set of photoprotective mechanisms to harmlessly dissipate excess energy as heat. To investigate these mechanisms, we study the primary antenna complex in green plants, light-harvesting complex II (LHCII), at the single-complex level. We use a single-molecule technique, the Anti-Brownian Electrokinetic trap, which enables simultaneous measurements of fluorescence intensity, lifetime, and spectra in solution. With this approach, including the first measurements of fluorescence lifetime on single LHCII complexes, we access the intrinsic conformational dynamics. In addition to an unquenched state, we identify two partially quenched states of LHCII. Our results suggest that there are at least two distinct quenching sites with different molecular compositions, meaning multiple dissipative pathways in LHCII. Furthermore, one of the quenched conformations significantly increases in relative population under environmental conditions mimicking high light.This material is based on work supported in part by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences, and Biosciences under Award Number DE-FG02-07ER15892 (to W.E.M) and by the Dutch organization for scientific research (NWO-ALW) via a Vici grant (to R.C.). R.v.G. and T.P.J.K. were supported by the Netherlands Organization for Sciences, Council of Chemical Sciences (NWO-CW) via a TOP-grant (700.58.305). R.v.G. was further supported by an Advanced Investigator grant from the European Research Council (no. 267333, PHOTPROT) and by the EU FP7 project PAPETS (GA 323901). R.v.G. gratefully acknowledges his Academy Professor grant from the Royal Netherlands Academy of Arts and Sciences (KNAW). T.P.J.K. was further supported by University of Pretoria’s Research Development Programme (grant no. A0W679). The authors would like to acknowledge the following fellowships: a Postdoctoral Fellowship from the Center for Molecular Analysis and Design at Stanford University (to G.S.S.-C.); a Kenneth and Nina Tai Stanford Graduate Fellowship (to H.-Y.Y.); and a Long Term Fellowship from EMBO (to M.G.).http://pubs.acs.org/journal/jpclcdhb2017Physic

Single-molecule identification of quenched and unquenched states of LHCII

In photosynthetic light harvesting, absorbed sunlight is converted to electron flow with near-unity quantum efficiency under low light conditions. Under high light conditions, plants avoid damage to their molecular machinery by activating a set of photoprotective mechanisms to harmlessly dissipate excess energy as heat. To investigate these mechanisms, we study the primary antenna complex in green plants, light-harvesting complex II (LHCII), at the single-complex level. We use a single-molecule technique, the Anti-Brownian Electrokinetic trap, which enables simultaneous measurements of fluorescence intensity, lifetime, and spectra in solution. With this approach, including the first measurements of fluorescence lifetime on single LHCII complexes, we access the intrinsic conformational dynamics. In addition to an unquenched state, we identify two partially quenched states of LHCII. Our results suggest that there are at least two distinct quenching sites with different molecular compositions, meaning multiple dissipative pathways in LHCII. Furthermore, one of the quenched conformations significantly increases in relative population under environmental conditions mimicking high light.This material is based on work supported in part by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, Division of Chemical Sciences, Geosciences, and Biosciences under Award Number DE-FG02-07ER15892 (to W.E.M) and by the Dutch organization for scientific research (NWO-ALW) via a Vici grant (to R.C.). R.v.G. and T.P.J.K. were supported by the Netherlands Organization for Sciences, Council of Chemical Sciences (NWO-CW) via a TOP-grant (700.58.305). R.v.G. was further supported by an Advanced Investigator grant from the European Research Council (no. 267333, PHOTPROT) and by the EU FP7 project PAPETS (GA 323901). R.v.G. gratefully acknowledges his Academy Professor grant from the Royal Netherlands Academy of Arts and Sciences (KNAW). T.P.J.K. was further supported by University of Pretoria’s Research Development Programme (grant no. A0W679). The authors would like to acknowledge the following fellowships: a Postdoctoral Fellowship from the Center for Molecular Analysis and Design at Stanford University (to G.S.S.-C.); a Kenneth and Nina Tai Stanford Graduate Fellowship (to H.-Y.Y.); and a Long Term Fellowship from EMBO (to M.G.).http://pubs.acs.org/journal/jpclcdhb2017Physic

Charting a course for chemistry

To mark the occasion of Nature Chemistry turning 10 years old, we asked scientists working in different areas of chemistry to tell us what they thought the most exciting, interesting or challenging aspects related to the development of their main field of research will be — here is what they said

Ultrabroadband 2D electronic spectroscopy as a tool for direct visualization of pathways of energy flow

Two-dimensional electronic spectroscopy (2DES) has emerged as an incisive tool for mapping out energy relaxation pathways in complex molecular systems by determining correlation maps between the excitation and emission frequencies. Its enhanced spectral as well as temporal resolution offer new insights into coupling and energy transfer between closely-spaced energy states, which are often hidden in a one-dimensional transient spectrum. However, a major drawback of the current 2DES technique is that the spectral window of detection is directly limited to the laser bandwidth used, which leads to an incomplete visualization of the full energy landscape of the system. As a solution to this limitation, we present an ultrabroadband 2DES apparatus utilizing a 8-fs, 185-nm bandwidth supercontinuum that covers the entire visible region. We demonstrate the utility of our setup by measuring the 2D spectra of laser dyes absorbing at different regions of the laser spectrum, and the major light-harvesting complex of spinach

Zeaxanthin independence of photophysics in light-harvesting complex II in a membrane environment

Green plants protect against photodamage by dissipating excess energy in a process called non-photochemical quenching (NPQ). In vivo, NPQ is activated by a drop in the luminal pH of the thylakoid membrane that triggers conformational changes of the antenna complexes, which activate quenching channels. The drop in pH also triggers de-epoxidation of violaxanthin, one of the carotenoids bound within the antenna complexes, into zeaxanthin, and so the amplitude of NPQ in vivo has been shown to increase in the presence of zeaxanthin. In vitro studies on light-harvesting complex II (LHCII), the major antenna complex in plants, compared different solubilization environments, which give rise to different levels of quenching and so partially mimic NPQ in vivo. However, in these studies both completely zeaxanthin-independent and zeaxanthin-dependent quenching have been reported, potentially due to the multiplicity of solubilization environments. Here, we characterize the zeaxanthin dependence of the photophysics in LHCII in a near-physiological membrane environment, which produces slightly enhanced quenching relative to detergent solubilization, the typical in vitro environment. The photophysical pathways of dark-adapted and in vitro de-epoxidized LHCIIs are compared, representative of the low-light and high-light conditions in vivo, respectively. The amplitude of quenching as well as the dissipative photophysics are unaffected by zeaxanthin at the level of individual LHCIIs, suggesting that zeaxanthin-dependent quenching is independent of the channels induced by the membrane. Furthermore, our results demonstrate that additional factors beyond zeaxanthin incorporation in LHCII are required for full development of NPQ

Identification of 1 distinct pH- and zeaxanthin-dependent quenching in LHCSR3 from chlamydomonas reinhardtii

Under high light, oxygenic photosynthetic organisms avoid photodamage by thermally dissipating absorbed energy, which is called non-photochemical quenching. In green algae, a chlorophyll and carotenoid-binding protein, light-harvesting complex stress17 related (LHCSR3), detects excess energy via a pH drop and serves as a quenching site. Using a combined in vivo and in vitro approach, we investigated quenching within LHCSR3 from Chlamydomonas reinhardtii. In vitro two distinct quenching processes, individually controlled by pH and zeaxanthin, were identified within LHCSR3. The pH21 dependent quenching was removed within a mutant LHCSR3 that lacks the residues that are protonated to sense the pH drop. Observation of quenching in zeaxanthin-enriched LHCSR3 even at neutral pH demonstrated zeaxanthin-dependent quenching, which also occurs in other light-harvesting complexes. Either pH- or zeaxanthin-dependent quenching prevented the formation of damaging reactive oxygen species, and thus the two quenching processes may together provide different induction and recovery kinetics for photoprotection in a changing environment.Human Frontier Science Program (Strasbourg, France) (Grant RGY0076)National Science Foundation (U.S.) (Grant HE-1740645

Ultrabroadband 2D electronic spectroscopy with high-speed, shot-to-shot detection

Two-dimensional electronic spectroscopy (2DES) is an incisive tool for disentangling excited state energies and dynamics in the condensed phase by directly mapping out the correlation between excitation and emission frequencies as a function of time. Despite its enhanced frequency resolution, the spectral window of detection is limited to the laser bandwidth, which has often hindered the visualization of full electronic energy relaxation pathways spread over the entire visible region. Here, we describe a high-sensitivity, ultrabroadband 2DES apparatus. We report a new combination of a simple and robust setup for increased spectral bandwidth and shot-to-shot detection. We utilize 8-fs supercontinuum pulses generated by gas filamentation spanning the entire visible region (450 – 800 nm), which allows for a simultaneous interrogation of electronic transitions over a 200-nm bandwidth, and an all-reflective interferometric delay system with angled nanopositioner stages achieves interferometric precision in coherence time control without introducing wavelength-dependent dispersion to the ultrabroadband spectrum. To address deterioration of detection sensitivity due to the inherent instability of ultrabroadband sources, we introduce a 5-kHz shot-to-shot, dual chopping acquisition scheme by combining a high-speed line-scan camera and two optical choppers to remove scatter contributions from the signal. Comparison of 2D spectra acquired by shot-to-shot detection and averaged detection shows a 15-fold improvement in the signal-to-noise ratio. This is the first direct quantification of detection sensitivity on a filamentation-based ultrabroadband 2DES apparatus

Ultrabroadband two-dimensional electronic spectroscopy reveals energy flow pathways in LHCII across the visible spectrum

We utilise ultrabroadband two-dimensional electronic spectroscopy to map out pathways of energy flow in LHCII across the entire visible region. In addition to the well-established, low-lying chlorophyll Qy bands, our results reveal additional pathways of energy relaxation on the higher-lying excited states involving the S2 energy levels of carotenoids, including ultrafast carotenoid-to-chlorophyll energy transfer on 90-150 fs timescales

Ultrabroadband two-dimensional electronic spectroscopy reveals energy flow pathways in LHCII across the visible spectrum

We utilise ultrabroadband two-dimensional electronic spectroscopy to map out pathways of energy flow in LHCII across the entire visible region. In addition to the well-established, low-lying chlorophyll Qy bands, our results reveal additional pathways of energy relaxation on the higher-lying excited states involving the S2 energy levels of carotenoids, including ultrafast carotenoid-to-chlorophyll energy transfer on 90-150 fs timescales