Publications by authors named "Gabriela S Schlau-Cohen"

33 Publications

Protein-Protein Interactions Induce pH-Dependent and Zeaxanthin-Independent Photoprotection in the Plant Light-Harvesting Complex, LHCII.

J Am Chem Soc 2021 Oct 14;143(42):17577-17586. Epub 2021 Oct 14.

Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.

Plants use energy from the sun yet also require protection against the generation of deleterious photoproducts from excess energy. Photoprotection in green plants, known as nonphotochemical quenching (NPQ), involves thermal dissipation of energy and is activated by a series of interrelated factors: a pH drop in the lumen, accumulation of the carotenoid zeaxanthin (Zea), and formation of arrays of pigment-containing antenna complexes. However, understanding their individual contributions and their interactions has been challenging, particularly for the antenna arrays, which are difficult to manipulate in vitro. Here, we achieved systematic and discrete control over the array size for the principal antenna complex, light-harvesting complex II, using near-native in vitro membranes called nanodiscs. Each of the factors had a distinct influence on the level of dissipation, which was characterized by measurements of fluorescence quenching and ultrafast chlorophyll-to-carotenoid energy transfer. First, an increase in array size led to a corresponding increase in dissipation; the dramatic changes in the chlorophyll dynamics suggested that this is due to an allosteric conformational change of the protein. Second, a pH drop increased dissipation but exclusively in the presence of protein-protein interactions. Third, no Zea dependence was identified which suggested that Zea regulates a distinct aspect of NPQ. Collectively, these results indicate that each factor provides a separate type of control knob for photoprotection, which likely enables a flexible and tunable response to solar fluctuations.
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http://dx.doi.org/10.1021/jacs.1c07385DOI Listing
October 2021

Concerted Differential Changes of Helical Dynamics and Packing upon Ligand Occupancy in a Bacterial Chemoreceptor.

ACS Chem Biol 2021 11 14;16(11):2472-2480. Epub 2021 Oct 14.

Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Ave, 6-225, Cambridge, Massachusetts 02139, United States.

Transmembrane receptors are central components of the chemosensory systems by which motile bacteria detect and respond to chemical gradients. An attractant bound to the receptor periplasmic domain generates conformational signals that regulate a histidine kinase interacting with its cytoplasmic domain. Ligand-induced signaling through the periplasmic and transmembrane domains of the receptor involves a piston-like helical displacement, but the nature of this signaling through the >200 Å four-helix coiled coil of the cytoplasmic domain had not yet been identified. We performed single-molecule Förster resonance energy transfer measurements on aspartate receptor homodimers inserted into native phospholipid bilayers enclosed in nanodiscs. The receptors were labeled with fluorophores at diagnostic positions near the middle of the cytoplasmic coiled coil. At these positions, we found that the two N-helices of the homodimer were more distant, that is, less tightly packed and more dynamic than the companion C-helix pair, consistent with previous deductions that the C-helices form a stable scaffold and the N-helices are dynamic. Upon ligand binding, the scaffold pair compacted further, while separation and dynamics of the dynamic pair increased. Thus, ligand binding had asymmetric effects on the two helical pairs, shifting mean distances in opposite directions and increasing the dynamics of one pair. We suggest that this reflects a conformational change in which differential alterations to the packing and dynamics of the two helical pairs are coupled. These coupled changes could represent a previously unappreciated mode of conformational signaling that may well occur in other coiled-coil signaling proteins.
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http://dx.doi.org/10.1021/acschembio.1c00576DOI Listing
November 2021

Spectrally-tunable femtosecond single-molecule pump-probe spectroscopy.

Opt Express 2021 Aug;29(18):28246-28256

Single-molecule spectroscopy has been extensively used to investigate heterogeneity in static and dynamic behaviors on millisecond and second timescales. More recently, single-molecule pump-probe spectroscopy emerged as a method to access heterogeneity on the femtosecond and picosecond timescales. Here, we develop a single-molecule pump-probe apparatus that is easily tunable across the visible region and demonstrate its utility on the widely-used fluorescent dye, Atto647N. A spectrally-independent, bimodal distribution of energetic relaxation time constants is found, where one peak corresponds to electronic dephasing (∼ 100 fs) and the other to intravibrational relaxation (∼ 300 fs). The bimodal nature indicates that relaxation within each individual molecule is dominated by only one of these processes. Both peaks of the distribution are narrow, suggesting little heterogeneity is present for either process. As illustrated here, spectrally-tunable single-molecule pump-probe spectroscopy will enable investigation of the heterogeneity in a wide range of biological and material systems.
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http://dx.doi.org/10.1364/OE.432995DOI Listing
August 2021

Ultrafast energy transfer between lipid-linked chromophores and plant light-harvesting complex II.

Phys Chem Chem Phys 2021 Sep 15;23(35):19511-19524. Epub 2021 Sep 15.

School of Physics and Astronomy, University of Leeds, Leeds LS2 9JT, UK.

Light-Harvesting Complex II (LHCII) is a membrane protein found in plant chloroplasts that has the crucial role of absorbing solar energy and subsequently performing excitation energy transfer to the reaction centre subunits of Photosystem II. LHCII provides strong absorption of blue and red light, however, it has minimal absorption in the green spectral region where solar irradiance is maximal. In a recent proof-of-principle study, we enhanced the absorption in this spectral range by developing a biohybrid system where LHCII proteins together with lipid-linked Texas Red (TR) chromophores were assembled into lipid membrane vesicles. The utility of these systems was limited by significant LHCII quenching due to protein-protein interactions and heterogeneous lipid structures. Here, we organise TR and LHCII into a lipid nanodisc, which provides a homogeneous, well-controlled platform to study the interactions between TR molecules and single LHCII complexes. Fluorescence spectroscopy determined that TR-to-LHCII energy transfer has an efficiency of at least 60%, resulting in a 262% enhancement of LHCII fluorescence in the 525-625 nm range, two-fold greater than in the previous system. Ultrafast transient absorption spectroscopy revealed two time constants of 3.7 and 128 ps for TR-to-LHCII energy transfer. Structural modelling and theoretical calculations indicate that these timescales correspond to TR-lipids that are loosely- or tightly-associated with the protein, respectively, with estimated TR-to-LHCII separations of ∼3.5 nm and ∼1 nm. Overall, we demonstrate that a nanodisc-based biohybrid system provides an idealised platform to explore the photophysical interactions between extrinsic chromophores and membrane proteins with potential applications in understanding more complex natural or artificial photosynthetic systems.
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http://dx.doi.org/10.1039/d1cp01628hDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8442836PMC
September 2021

Membrane-dependent heterogeneity of LHCII characterized using single-molecule spectroscopy.

Biophys J 2021 08 30;120(15):3091-3102. Epub 2021 Jun 30.

Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts. Electronic address:

In green plants, light harvesting complex of Photosystem II (LHCII) absorbs and transports excitation energy toward the photosynthetic reaction centers and serves as a site for energy-dependent nonphotochemical quenching (qE), the photoprotective dissipation of energy as heat. LHCII is thought to activate dissipation through conformational changes that change the photophysical behaviors. Understanding this balance requires a characterization of how the conformations of LHCII, and thus its photophysics, are influenced by individual factors within the membrane environment. Here, we used ensemble and single-molecule fluorescence to characterize the excited-state lifetimes and switching kinetics of LHCII embedded in nanodisc- and liposome-based model membranes of various sizes and lipid compositions. As the membrane area decreased, the quenched population and the rate of conformational dynamics both increased because of interactions with other proteins, the aqueous solution, and/or disordered lipids. Although the conformational states and dynamics were similar in both thylakoid and asolectin lipids, photodegradation increased with thylakoid lipids, likely because of their charge and pressure properties. Collectively, these findings demonstrate the ability of membrane environments to tune the conformations and photophysics of LHCII.
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http://dx.doi.org/10.1016/j.bpj.2021.06.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8390968PMC
August 2021

Identification of distinct pH- and zeaxanthin-dependent quenching in LHCSR3 from .

Elife 2021 Jan 15;10. Epub 2021 Jan 15.

Department of Chemistry, Massachusetts Institute of Technology, Cambridge, United States.

Under high light, oxygenic photosynthetic organisms avoid photodamage by thermally dissipating absorbed energy, which is called nonphotochemical quenching. In green algae, a chlorophyll and carotenoid-binding protein, light-harvesting complex stress-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 In vitro two distinct quenching processes, individually controlled by pH and zeaxanthin, were identified within LHCSR3. The pH-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.
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http://dx.doi.org/10.7554/eLife.60383DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7864637PMC
January 2021

Phosphorylation-Dependent Conformations of the Disordered Carboxyl-Terminus Domain in the Epidermal Growth Factor Receptor.

J Phys Chem Lett 2020 Dec 12;11(23):10037-10044. Epub 2020 Nov 12.

Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.

The epidermal growth factor receptor (EGFR), a receptor tyrosine kinase, regulates basic cellular functions and is a major target for anticancer therapeutics. The carboxyl-terminus domain is a disordered region of EGFR that contains the tyrosine residues, which undergo autophosphorylation followed by docking of signaling proteins. Local phosphorylation-dependent secondary structure has been identified and is thought to be associated with the signaling cascade. Deciphering and distinguishing the overall conformations, however, have been challenging because of the disordered nature of the carboxyl-terminus domain and resultant lack of well-defined three-dimensional structure for most of the domain. We investigated the overall conformational states of the isolated EGFR carboxyl-terminus domain using single-molecule Förster resonance energy transfer and coarse-grained simulations. Our results suggest that electrostatic interactions between charged residues emerge within the disordered domain upon phosphorylation, producing a looplike conformation. This conformation may enable binding of downstream signaling proteins and potentially reflect a general mechanism in which electrostatics transiently generate functional architectures in disordered regions of a well-folded protein.
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http://dx.doi.org/10.1021/acs.jpclett.0c02327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8063277PMC
December 2020

Identification of Nonradiative Decay Pathways in Cy3.

J Phys Chem Lett 2020 Jul 12;11(13):5000-5007. Epub 2020 Jun 12.

Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, United States.

Photoexcited fluorescent markers are extensively used in spectroscopy, imaging, and analysis of biological systems. The performance of fluorescent markers depends on high levels of emission, which are limited by competing nonradiative decay pathways. Small-molecule fluorescent dyes have been increasingly used as markers due to their high and stable emission. Despite their prevalence, the nonradiative decay pathways of these dyes have not been determined. Here, we investigate these pathways for a widely used indocarbocyanine dye, Cy3, using transient grating spectroscopy. We identify a nonradiative decay pathway via a previously unknown dark state formed within ∼1 ps of photoexcitation. Our experiments, in combination with electronic structure calculations, suggest that the generation of the dark state is mediated by picosecond vibrational mode coupling, likely via a conical intersection. We further identify the vibrational modes, and thus structural elements, responsible for the formation and dynamics of the dark state, providing insight into suppressing nonradiative decay pathways in fluorescent markers such as Cy3.
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http://dx.doi.org/10.1021/acs.jpclett.0c01201DOI Listing
July 2020

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

Biochim Biophys Acta Bioenerg 2020 06 20;1861(5-6):148115. Epub 2020 Mar 20.

Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.bbabio.2019.148115DOI Listing
June 2020

Observation of dissipative chlorophyll-to-carotenoid energy transfer in light-harvesting complex II in membrane nanodiscs.

Nat Commun 2020 03 10;11(1):1295. Epub 2020 Mar 10.

Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA, 02139, USA.

Plants prevent photodamage under high light by dissipating excess energy as heat. Conformational changes of the photosynthetic antenna complexes activate dissipation by leveraging the sensitivity of the photophysics to the protein structure. The mechanisms of dissipation remain debated, largely due to two challenges. First, because of the ultrafast timescales and large energy gaps involved, measurements lacked the temporal or spectral requirements. Second, experiments have been performed in detergent, which can induce non-native conformations, or in vivo, where contributions from homologous antenna complexes cannot be disentangled. Here, we overcome both challenges by applying ultrabroadband two-dimensional electronic spectroscopy to the principal antenna complex, LHCII, in a near-native membrane. Our data provide evidence that the membrane enhances two dissipative pathways, one of which is a previously uncharacterized chlorophyll-to-carotenoid energy transfer. Our results highlight the sensitivity of the photophysics to local environment, which may control the balance between light harvesting and dissipation in vivo.
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http://dx.doi.org/10.1038/s41467-020-15074-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7064482PMC
March 2020

Comparison of the Energy-Transfer Rates in Structural and Spectral Variants of the B800-850 Complex from Purple Bacteria.

J Phys Chem B 2020 02 14;124(8):1460-1469. Epub 2020 Feb 14.

Department of Chemistry , Massachusetts Institute of Technology , Cambridge , Massachusetts 02139 , United States.

Photosynthetic light harvesting can occur with a remarkable near-unity quantum efficiency. The B800-850 complex, also known as light-harvesting complex 2 (LH2), is the primary light-harvesting complex in purple bacteria and has been extensively studied as a model system. The bacteriochlorophylls of the B800-850 complex are organized into two concentric rings, known as the B800 and B850 rings. However, depending on the species and growth conditions, the number of constituent subunits, the pigment geometry, and the absorption energies vary. While the dynamics of some B800-850 variants have been exhaustively characterized, others have not been measured. Furthermore, a direct and simultaneous comparison of how both structural and spectral differences between variants affect these dynamics has not been performed. In this work, we utilize ultrafast transient absorption measurements to compare the B800 to B850 energy-transfer rates in the B800-850 complex as a function of the number of subunits, geometry, and absorption energies. The nonameric B800-850 complex from is 40% faster than the octameric B800-850 complex from , consistent with structure-based predictions. In contrast, the blue-shifted B800-820 complex from is only 20% faster than the B800-850 complex from despite an increase in the spectral overlap between the rings that would be expected to produce a larger increase in the energy-transfer rate. These measurements support current models that contain dark, higher-lying excitonic states to bridge the energy gap between rings, thereby maintaining similar energy-transfer dynamics. Overall, these results demonstrate that energy-transfer dynamics in the B800-850 complex are robust to the spectral and structural variations between species used to optimize energy capture and flow in purple bacteria.
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http://dx.doi.org/10.1021/acs.jpcb.9b11899DOI Listing
February 2020

Microsecond and millisecond dynamics in the photosynthetic protein LHCSR1 observed by single-molecule correlation spectroscopy.

Proc Natl Acad Sci U S A 2019 06 17;116(23):11247-11252. Epub 2019 May 17.

Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139;

Biological systems are subjected to continuous environmental fluctuations, and therefore, flexibility in the structure and function of their protein building blocks is essential for survival. Protein dynamics are often local conformational changes, which allows multiple dynamical processes to occur simultaneously and rapidly in individual proteins. Experiments often average over these dynamics and their multiplicity, preventing identification of the molecular origin and impact on biological function. Green plants survive under high light by quenching excess energy, and Light-Harvesting Complex Stress Related 1 (LHCSR1) is the protein responsible for quenching in moss. Here, we expand an analysis of the correlation function of the fluorescence lifetime by improving the estimation of the lifetime states and by developing a multicomponent model correlation function, and we apply this analysis at the single-molecule level. Through these advances, we resolve previously hidden rapid dynamics, including multiple parallel processes. By applying this technique to LHCSR1, we identify and quantitate parallel dynamics on hundreds of microseconds and tens of milliseconds timescales, likely at two quenching sites within the protein. These sites are individually controlled in response to fluctuations in sunlight, which provides robust regulation of the light-harvesting machinery. Considering our results in combination with previous structural, spectroscopic, and computational data, we propose specific pigments that serve as the quenching sites. These findings, therefore, provide a mechanistic basis for quenching, illustrating the ability of this method to uncover protein function.
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http://dx.doi.org/10.1073/pnas.1821207116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6561297PMC
June 2019

Impact of the lipid bilayer on energy transfer kinetics in the photosynthetic protein LH2.

Chem Sci 2018 Mar 9;9(12):3095-3104. Epub 2018 Feb 9.

Department of Chemistry , Massachusetts Institute of Technology , Cambridge , MA 02139 , USA . Email:

Photosynthetic purple bacteria convert solar energy to chemical energy with near unity quantum efficiency. The light-harvesting process begins with absorption of solar energy by an antenna protein called Light-Harvesting Complex 2 (LH2). Energy is subsequently transferred within LH2 and then through a network of additional light-harvesting proteins to a central location, termed the reaction center, where charge separation occurs. The energy transfer dynamics of LH2 are highly sensitive to intermolecular distances and relative organizations. As a result, minor structural perturbations can cause significant changes in these dynamics. Previous experiments have primarily been performed in two ways. One uses non-native samples where LH2 is solubilized in detergent, which can alter protein structure. The other uses complex membranes that contain multiple proteins within a large lipid area, which make it difficult to identify and distinguish perturbations caused by protein-protein interactions and lipid-protein interactions. Here, we introduce the use of the biochemical platform of model membrane discs to study the energy transfer dynamics of photosynthetic light-harvesting complexes in a near-native environment. We incorporate a single LH2 from into membrane discs that provide a spectroscopically amenable sample in an environment more physiological than detergent but less complex than traditional membranes. This provides a simplified system to understand an individual protein and how the lipid-protein interaction affects energy transfer dynamics. We compare the energy transfer rates of detergent-solubilized LH2 with those of LH2 in membrane discs using transient absorption spectroscopy and transient absorption anisotropy. For one key energy transfer step in LH2, we observe a 30% enhancement of the rate for LH2 in membrane discs compared to that in detergent. Based on experimental results and theoretical modeling, we attribute this difference to tilting of the peripheral bacteriochlorophyll in the B800 band. These results highlight the importance of well-defined systems with near-native membrane conditions for physiologically-relevant measurements.
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http://dx.doi.org/10.1039/c7sc04814aDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5914429PMC
March 2018

Single-Molecule Fluorescence Detection of the Epidermal Growth Factor Receptor in Membrane Discs.

Biochemistry 2019 01 6;58(4):286-294. Epub 2018 Apr 6.

Department of Chemistry , Massachusetts Institute of Technology , 77 Massachusetts Avenue , Cambridge , Massachusetts 02139 , United States.

The epidermal growth factor receptor (EGFR) is critical to normal cellular signaling pathways. Moreover, it has been implicated in a range of pathologies, including cancer. As a result, it is the primary target of many anticancer drugs. One limitation to the design and development of these drugs has been the lack of molecular-level information about the interactions and conformational dynamics of EGFR. To overcome this limitation, this work reports the construction and characterization of functional, fluorescently labeled, and full-length EGFR in model membrane nanolipoprotein particles (NLPs) for in vitro fluorescence studies. To demonstrate the utility of the system, we investigate ATP-EGFR interactions. We observe that ATP binds at the catalytic site providing a means to measure a range of distances between the catalytic site and the C-terminus via Förster resonance energy transfer (FRET). These ATP-based experiments suggest a range of conformations of the C-terminus that may be a function of the phosphorylation state for EGFR. This work is a proof-of-principle demonstration of single-molecule studies as a noncrystallographic assay for EGFR interactions in real-time and under near-physiological conditions. The diverse nature of EGFR interactions means that new tools at the molecular level have the potential to significantly enhance our understanding of receptor pathology and are of utmost importance for cancer-related drug discovery.
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http://dx.doi.org/10.1021/acs.biochem.8b00089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6173994PMC
January 2019

Programmed coherent coupling in a synthetic DNA-based excitonic circuit.

Nat Mater 2018 02 13;17(2):159-166. Epub 2017 Nov 13.

Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.

Natural light-harvesting systems spatially organize densely packed chromophore aggregates using rigid protein scaffolds to achieve highly efficient, directed energy transfer. Here, we report a synthetic strategy using rigid DNA scaffolds to similarly program the spatial organization of densely packed, discrete clusters of cyanine dye aggregates with tunable absorption spectra and strongly coupled exciton dynamics present in natural light-harvesting systems. We first characterize the range of dye-aggregate sizes that can be templated spatially by A-tracts of B-form DNA while retaining coherent energy transfer. We then use structure-based modelling and quantum dynamics to guide the rational design of higher-order synthetic circuits consisting of multiple discrete dye aggregates within a DX-tile. These programmed circuits exhibit excitonic transport properties with prominent circular dichroism, superradiance, and fast delocalized exciton transfer, consistent with our quantum dynamics predictions. This bottom-up strategy offers a versatile approach to the rational design of strongly coupled excitonic circuits using spatially organized dye aggregates for use in coherent nanoscale energy transport, artificial light-harvesting, and nanophotonics.
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http://dx.doi.org/10.1038/nmat5033DOI Listing
February 2018

Photophysics of J-Aggregate-Mediated Energy Transfer on DNA.

J Phys Chem Lett 2017 Dec 16;8(23):5827-5833. Epub 2017 Nov 16.

Energy Frontier Research Center for Excitonics, ‡Department of Biological Engineering, and §Department of Chemistry, Massachusetts Institute of Technology , 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States.

Achieving nanoscale spatial and electronic control over the formation of dye aggregates is a major synthetic challenge due to their typically inhomogeneous self-assembly, which limits control over their higher-order organization. To address these challenges, synthetic DNA-templated pseudoisocyanine (PIC) J-aggregates were recently introduced. However, the dependence of the photophysics of the superradiant exciton on the underlying DNA template length and the impact of static disorder on energy transfer through these PIC J-aggregates remain unknown. We examine the delocalization length progression of superradiant PIC excitons by varying the length of poly-A DNA tracts that template PIC J-aggregates. We then investigate the energy-transfer efficiency from PIC J-aggregates with DNA duplex template length, which we found to be limited by static disorder. Utilizing the self-assembled and selective formation of superradiant excitons on DNA provides a platform to determine the function of delocalized excitons in the context of nanoscale energy transport.
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http://dx.doi.org/10.1021/acs.jpclett.7b01898DOI Listing
December 2017

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

Opt Express 2017 Aug;25(16):18950-18962

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.
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http://dx.doi.org/10.1364/OE.25.018950DOI Listing
August 2017

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

Nat Chem 2017 08 17;9(8):772-778. Epub 2017 Jul 17.

Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.

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.
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http://dx.doi.org/10.1038/nchem.2818DOI Listing
August 2017

Single-Molecule Fluorescence Spectroscopy of Photosynthetic Systems.

Chem Rev 2017 01 10;117(2):860-898. Epub 2017 Jan 10.

Department of Chemistry, Massachusetts Institute of Technology , 77 Massachusetts Avenue, Cambridge Massachusetts 02139, United States.

Photosynthesis begins when a network of pigment-protein complexes captures solar energy and transports it to the reaction center, where charge separation occurs. When necessary (under low light conditions), photosynthetic organisms perform this energy transport and charge separation with near unity quantum efficiency. Remarkably, this high efficiency is maintained under physiological conditions, which include thermal fluctuations of the pigment-protein complexes and changing local environments. These conditions introduce multiple types of heterogeneity in the pigment-protein complexes, including structural heterogeneity, energetic heterogeneity, and functional heterogeneity. Understanding how photosynthetic light-harvesting functions in the face of these fluctuations requires understanding this heterogeneity, which, in turn, requires characterization of individual pigment-protein complexes. Single-molecule spectroscopy has the power to probe individual complexes. In this review, we present an overview of the common techniques for single-molecule fluorescence spectroscopy applied to photosynthetic systems and describe selected experiments on these systems. We discuss how these experiments provide a new understanding of the impact of heterogeneity on light harvesting and thus how these systems are optimized to capture sunlight under physiological conditions.
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http://dx.doi.org/10.1021/acs.chemrev.6b00195DOI Listing
January 2017

Single-Molecule Identification of Quenched and Unquenched States of LHCII.

J Phys Chem Lett 2015 Mar 23;6(5):860-7. Epub 2015 Feb 23.

†Department of Chemistry, Stanford University, Stanford, California 94305, United States.

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.
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http://dx.doi.org/10.1021/acs.jpclett.5b00034DOI Listing
March 2015

Insights into the structural changes occurring upon photoconversion in the orange carotenoid protein from broadband two-dimensional electronic spectroscopy.

J Phys Chem B 2014 May 9;118(20):5382-9. Epub 2014 May 9.

Applied Science and Technology Graduate Group, University of California , Berkeley, California 94720, United States.

Carotenoids play an essential role in photoprotection, interacting with other pigments to safely dissipate excess absorbed energy as heat. In cyanobacteria, the short time scale photoprotective mechanisms involve the photoactive orange carotenoid protein (OCP), which binds a single carbonyl carotenoid. Blue-green light induces the photoswitching of OCP from its ground state form (OCPO) to a metastable photoproduct (OCPR). OCPR can bind to the phycobilisome antenna and induce fluorescence quenching. The photoswitching is accompanied by structural and functional changes at the level of the protein and of the bound carotenoid. Here, we use broadband two-dimensional electronic spectroscopy to study the differences in excited state dynamics of the carotenoid in the two forms of OCP. Our results provide insight into the origin of the pronounced vibrational lineshape and oscillatory dynamics observed in linear absorption and 2D electronic spectroscopy of OCPO and the large inhomogeneous broadening in OCPR, with consequences for the chemical function of the two forms.
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http://dx.doi.org/10.1021/jp502120hDOI Listing
May 2014

Single-molecule spectroscopy reveals photosynthetic LH2 complexes switch between emissive states.

Proc Natl Acad Sci U S A 2013 Jul 17;110(27):10899-903. Epub 2013 Jun 17.

Department of Chemistry, Stanford University, Stanford, CA 94305, USA.

Photosynthetic organisms flourish under low light intensities by converting photoenergy to chemical energy with near unity quantum efficiency and under high light intensities by safely dissipating excess photoenergy and deleterious photoproducts. The molecular mechanisms balancing these two functions remain incompletely described. One critical barrier to characterizing the mechanisms responsible for these processes is that they occur within proteins whose excited-state properties vary drastically among individual proteins and even within a single protein over time. In ensemble measurements, these excited-state properties appear only as the average value. To overcome this averaging, we investigate the purple bacterial antenna protein light harvesting complex 2 (LH2) from Rhodopseudomonas acidophila at the single-protein level. We use a room-temperature, single-molecule technique, the anti-Brownian electrokinetic trap, to study LH2 in a solution-phase (nonperturbative) environment. By performing simultaneous measurements of fluorescence intensity, lifetime, and spectra of single LH2 complexes, we identify three distinct states and observe transitions occurring among them on a timescale of seconds. Our results reveal that LH2 complexes undergo photoactivated switching to a quenched state, likely by a conformational change, and thermally revert to the ground state. This is a previously unobserved, reversible quenching pathway, and is one mechanism through which photosynthetic organisms can adapt to changes in light intensities.
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http://dx.doi.org/10.1073/pnas.1310222110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3704035PMC
July 2013

Determination of Excited-State Energies and Dynamics in the B Band of the Bacterial Reaction Center with 2D Electronic Spectroscopy.

J Phys Chem Lett 2012 Sep 22;3(17):2487-92. Epub 2012 Aug 22.

§Physical Biosciences Division, Lawrence Berkeley National Lab, Berkeley, California, United States.

Photosynthetic organisms convert photoenergy to chemical energy with near-unity quantum efficiency. This occurs through charge transfer in the reaction center, which consists of two branches of pigments. In bacteria, both branches are energy-transfer pathways, but only one is also an electron transfer pathway. One barrier to a full understanding of the asymmetry is that the two branches contain excited states close in energy that produce overlapping spectroscopic peaks. We apply polarization-dependent, 2D electronic spectroscopy to the B band of the oxidized bacterial reaction center. The spectra reveal two previously unresolved peaks, corresponding to excited states localized on each of the two branches. Furthermore, a previously unknown interaction between these two states is observed on a time scale of ∼100 fs. This may indicate an alternative pathway to electron transfer for the oxidized reaction center and thus may be a mechanism to prevent energy from becoming trapped in local minima.
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http://dx.doi.org/10.1021/jz300841uDOI Listing
September 2012

Elucidation of the timescales and origins of quantum electronic coherence in LHCII.

Nat Chem 2012 Mar 25;4(5):389-95. Epub 2012 Mar 25.

Department of Chemistry, University of California, Berkeley, California 94720, USA.

Photosynthetic organisms harvest sunlight with near unity quantum efficiency. The complexity of the electronic structure and energy transfer pathways within networks of photosynthetic pigment-protein complexes often obscures the mechanisms behind the efficient light-absorption-to-charge conversion process. Recent experiments, particularly using two-dimensional spectroscopy, have detected long-lived quantum coherence, which theory suggests may contribute to the effectiveness of photosynthetic energy transfer. Here, we present a new, direct method to access coherence signals: a coherence-specific polarization sequence, which isolates the excitonic coherence features from the population signals that usually dominate two-dimensional spectra. With this polarization sequence, we elucidate coherent dynamics and determine the overall measurable lifetime of excitonic coherence in the major light-harvesting complex of photosystem II. Coherence decays on two distinct timescales of 47 fs and ~800 fs. We present theoretical calculations to show that these two timescales are from weakly and moderately strongly coupled pigments, respectively.
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http://dx.doi.org/10.1038/nchem.1303DOI Listing
March 2012

Design principles of photosynthetic light-harvesting.

Faraday Discuss 2012 ;155:27-41; discussion 103-14

Department of Chemistry, University of California, Berkeley, CA 94720, USA.

Photosynthetic organisms are capable of harvesting solar energy with near unity quantum efficiency. Even more impressively, this efficiency can be regulated in response to the demands of photosynthetic reactions and the fluctuating light-levels of natural environments. We discuss the distinctive design principles through which photosynthetic light-harvesting functions. These emergent properties of photosynthesis appear both within individual pigment-protein complexes and in how these complexes integrate to produce a functional, regulated apparatus that drives downstream photochemistry. One important property is how the strong interactions and resultant quantum coherence, produced by the dense packing of photosynthetic pigments, provide a tool to optimize for ultrafast, directed energy transfer. We also describe how excess energy is quenched to prevent photodamage under high-light conditions, which we investigate through theory and experiment. We conclude with comments on the potential of using these features to improve solar energy devices.
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http://dx.doi.org/10.1039/c1fd00078kDOI Listing
April 2012

Spectroscopic elucidation of uncoupled transition energies in the major photosynthetic light-harvesting complex, LHCII.

Proc Natl Acad Sci U S A 2010 Jul 9;107(30):13276-81. Epub 2010 Jul 9.

Department of Chemistry, University of California, Berkeley, CA 94720, USA.

Electrostatic couplings between chromophores in photosynthetic pigment-protein complexes, and interactions of pigments with the surrounding protein environment, produce a complicated energy landscape of delocalized excited states. The resultant electronic structure absorbs light and gives rise to energy transfer steps that direct the excitation toward a site of charge separation with near unity quantum efficiency. Knowledge of the transition energies of the uncoupled chromophores is required to describe how the wave functions of the individual pigments combine to form this manifold of delocalized excited states that effectively harvests light energy. In an investigation of the major light-harvesting complex of photosystem II (LHCII), we develop a method based on polarized 2D electronic spectroscopy to experimentally access the energies of the S(0)-S(1) transitions in the chromophore site basis. Rotating the linear polarization of the incident laser pulses reveals previously hidden off-diagonal features. We exploit the polarization dependence of energy transfer peaks to find the angles between the excited state transition dipole moments. We show that these angles provide a spectroscopic method to directly inform on the relationship between the delocalized excitons and the individual chlorophylls through the site energies of the uncoupled chromophores.
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http://dx.doi.org/10.1073/pnas.1006230107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2922122PMC
July 2010

Quantum coherence and its interplay with protein environments in photosynthetic electronic energy transfer.

Phys Chem Chem Phys 2010 Jul 12;12(27):7319-37. Epub 2010 Jun 12.

Department of Chemistry, University of California, Berkeley, CA 94720, USA.

Recent experiments suggest that electronic energy transfer in photosynthetic pigment-protein complexes involves long-lived quantum coherence among electronic excitations of pigments. [Engel et al., Nature, 2007, 446, 782-786.] The observation has led to the suggestion that quantum coherence might play a significant role in achieving the remarkable efficiency of photosynthetic light harvesting. At the same time, the observation has raised questions regarding the role of the surrounding protein in protecting the quantum coherence. In this Perspective, we provide an overview of recent experimental and theoretical investigations of photosynthetic electronic energy transfer paying particular attention to the underlying mechanisms of long-lived quantum coherence and its non-Markovian interplay with the protein environment.
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http://dx.doi.org/10.1039/c003389hDOI Listing
July 2010

Quantum coherence enabled determination of the energy landscape in light-harvesting complex II.

J Phys Chem B 2009 Dec;113(51):16291-5

The near-unity efficiency of energy transfer in photosynthesis makes photosynthetic light-harvesting complexes a promising avenue for developing new renewable energy technologies. Knowledge of the energy landscape of these complexes is essential in understanding their function, but its experimental determination has proven elusive. Here, the observation of quantum coherence using two-dimensional electronic spectroscopy is employed to directly measure the 14 lowest electronic energy levels in light-harvesting complex II (LHCII), the most abundant antenna complex in plants containing approximately 50% of the world's chlorophyll. We observe that the electronically excited states are relatively evenly distributed, highlighting an important design principle of photosynthetic complexes that explains the observed ultrafast intracomplex energy transfer in LHCII.
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http://dx.doi.org/10.1021/jp908300cDOI Listing
December 2009
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