Preparation, Purification, and Crystallization of Purple Bacteria Antenna Complexes

Cogdell, Richard J.; Hawthornthwaite, A. M.

doi:10.1016/b978-0-12-208661-8.50008-5

Cited by 35 publications

(31 citation statements)

References 12 publications

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“…Purified LH2 complexes of Rps. acidophila were prepared as described previously (35). The stock solution of LH2 suspended in 20 mM Tris·HCl (pH 8.0) and 0.1% lauryldimethylamine oxide was kept at −80°C and thawed immediately before the experiments.…”

Section: Methodsmentioning

confidence: 99%

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

Schlau‐Cohen¹,

Wang

Southall

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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126

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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 antiBrownian 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.photosynthesis | purple bacteria | fluorescence spectroscopy I n photosynthetic light harvesting, networks of antenna pigmentprotein complexes (PPCs) absorb sunlight, then efficiently transport the excitation through these networks to the reaction center, a PPC dedicated to charge separation (1, 2). Photosynthetic systems can complete this process both with near unity quantum efficiency and also function at light levels that provide photoenergy in excess of the capacity of downstream photochemistry. This is accomplished through mechanisms that dissipate harmful by-products produced by unused photoenergy (2, 3). The absorption, energy transport, and dissipation properties are governed by the balance of pigment-pigment and pigment-protein couplings (4). However, the molecular machinery responsible for this balance, and for the couplings themselves, is still poorly understood. This is because the couplings are highly sensitive to intermolecular distances. As a result of this sensitivity, the light-harvesting properties vary drastically among individual PPCs, because of small differences in protein conformation, and vary drastically even within a single PPC over time, because of protein fluctuations (2). In ensemble measurements, these drastic variations appear solely as a static, average value. Therefore, only through single-molecule spectroscopy can we investigate the photodynamics of individual PPCs, specifically how the absorption, energy transport, and dissipation of each change with time. We ...

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Section: Methodsmentioning

confidence: 99%

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

Schlau‐Cohen¹,

Wang

Southall

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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126

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“…terized by X-ray crystallography and electron microscopy (Welte et al, 1985;Cogdell & Hawthornthwaite, 1993;Germeroth et al, 1993;Karrasch et al, 1995), no structure has yet been publ i~h e d . ~ We attempted to predict the structure of LH-I1 of Rs.…”

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confidence: 99%

Predicting the structure of the light-harvesting complex II ofrhodospirillum molischianum

Hamer

et al. 1995

Protein Sci.

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We attempted to predict through computer modeling the structure of the light-harvesting complex II (LH-Ii) of Rhodospirillum molischianum, before the impending publication of the structure of a homologous protein solved by means of X-ray diffraction. The protein studied is an integral membrane protein of 16 independent polypeptides, 8 a-apoproteins and 8 @-apoproteins, which aggregate and bind to 24 bacteriochlorophyll-a's and 12 lycopenes. Available diffraction data of a crystal of the protein, which could not be phased due to a lack of heavy metal derivatives, served to test the predicted structure, guiding the search. In order to determine the secondary structure, hydropathy analysis was performed to identify the putative transmembrane segments and multiple sequence alignment propensity analyses were used to pinpoint the exact sites of the 20-residue-long transmembrane segment and the 4-residue-long terminal sequence at both ends, which were independently verified and improved by homology modeling. A consensus assignment for the secondary structure was derived from a combination of all the prediction methods used. Three-dimensional structures for the cy-and the @-apoprotein were built by comparative modeling. The resulting tertiary structures are combined, using X-PLOR, into an a@ dimer pair with bacteriochlorophyll-a's attached under constraints provided by site-directed mutagenesis and spectral data. The a@ dimer pairs were then aggregated into a quaternary structure through further molecular dynamics simulations and energy minimization. The structure of LH-I1 so determined is an octamer of a@ heterodimers forming a ring with a diameter of 70 A.

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“…acidophila (strain 10050) were isolated and purified as described previously 37 . After purification the LH2 complexes were aliquoted and stored at - 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 …”

Section: Methodsmentioning

confidence: 99%

Multi-Level, Multi Time-Scale Fluorescence Intermittency of Photosynthetic LH2 Complexes: A Precursor of Non-Photochemical Quenching?

et al. 2015

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The light harvesting complex LH2 is a chromoprotein that is an ideal system for studying protein dynamics via the spectral fluctuations of the emission of its intrinsic chromophores. We have immobilized these complexes in a polymer film and studied the fluctuations of the fluorescence intensity from individual complexes over 9 orders of magnitude in time. Combining time-tagged detection of single photons with a change-point analysis has allowed the unambigeous identification of the various intensity levels due to the huge statistical basis of the data set. We propose that the observed intensity level fluctuations reflect conformational changes of the protein backbone that might be a precursor of the mechanism from which nonphotochemical quenching of higher plants has evolved.

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Preparation, Purification, and Crystallization of Purple Bacteria Antenna Complexes

Cited by 35 publications

References 12 publications

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

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

Predicting the structure of the light-harvesting complex II ofrhodospirillum molischianum

Multi-Level, Multi Time-Scale Fluorescence Intermittency of Photosynthetic LH2 Complexes: A Precursor of Non-Photochemical Quenching?

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