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Regulating the Energy Flow in a Cyanobacterial Light Harvesting Antenna Complex Ido Eisenberg, Felipe Caycedo-Soler, Dvir Harris, Shira Yochelis, Susana F. Huelga, Martin B. Plenio, Noam Adir, Nir Keren, and Yossi Paltiel J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b10590 • Publication Date (Web): 25 Jan 2017 Downloaded from http://pubs.acs.org on January 26, 2017

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The Journal of Physical Chemistry B is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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The Journal of Physical Chemistry

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Regulating the Energy Flow in a Cyanobacterial 7 8 9 10 1

Light Harvesting Antenna Complex 12 13 14 15 16

Ido Eisenberg1*, Felipe Caycedo-Soler2, Dvir Harris3, Shira Yochelis1, Susana F. Huelga2, 17 18

Martin B. Plenio2, Noam Adir3, Nir Keren4, Yossi Paltiel1* 19 20 21 2

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Applied Physics Department and The Center for Nano-Science and Nano-Technology, The

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Hebrew University of Jerusalem, Jerusalem, 9190401 Israel 25 26 27 2

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Institute of Theoretical Physics, Ulm University, Albert Einstein Alle 11, 89069 Ulm, Germany

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Schulich Faculty of Chemistry, Technion - Israel Institute of Technology, Haifa, 32000 Israel

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Department of Plant and Environmental Sciences, Alexander Silberman Institute of Life

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Sciences, Givat Ram, The Hebrew University of Jerusalem, Jerusalem 9190401, Israel 38 39 40 41 42 43 4 45 46 47 48 49 50 51 52 53 54 5 56 57 58 59 60 ACS Paragon Plus Environment

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The Journal of Physical Chemistry

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ABSTRACT 4 5 7

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Photosynthetic organisms harvest light energy, utilizing the absorption and energy transfer 9

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properties of protein-bound chromophores. Controlling the harvesting efficiency is critical for 10 1

the optimal function of the photosynthetic apparatus. Here, we show that cyanobacterial light12 14

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harvesting antenna may be able to regulate the flow of energy in order to switch reversibly from 16

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efficient energy conversion to photo-protective quenching via a structural change. We isolated 17 19

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cyanobacterial light harvesting proteins, phycocyanin and allophycocyanin, and measured their 21

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optical properties in solution and in an aggregated-desiccated state. The results indicate that 2 23

energy band structures are changed, generating a switch between two modes of operation: 24 26

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exciton transfer and quenching; achieved without dedicated carotenoid quenchers. This 28

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flexibility can contribute greatly to the large dynamic range of cyanobacterial light harvesting 29 30

systems. 31 32 3 34 35 36 38

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INTRODUCTION 39 41

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Photosynthetic organisms are able to thrive in environments in which the light intensities are 42 43

constantly changing. This is made possible by the existence of molecular mechanisms that 4 46

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provide the dynamic range required for both extremely efficient excitation energy transfer (EET) 48

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to drive light-chemical energy conversion, and to quench energy when photochemistry is 49 50

blocked or becomes rate-limiting.1–3 In the green photosynthetic lineage (green algae and 53

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vascular plants) the EET process utilizes chlorophylls and energy quenching is performed mainly 54 5 56 57 58 59 60 ACS Paragon Plus Environment

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by carotenoids.1,4–6 Balancing these two processes under changing external conditions is 5

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achieved by adaptation mechanisms that include structural and conformational changes.1,7–10 6 8

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Within their antenna complex, the phycobilisome (PBS), cyanobacteria utilize linear tetrapyrrole 10

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(bilin) chromophores covalently bound to dedicated phycobiliproteins (PBPs) for EET.7,11–13 In 1 12

some species, under high light conditions, the orange carotenoid protein (OCP) is activated to 13 14

promote quenching.14–16 PBPs themselves do not contain any carotenoids. 17

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The PBS is composed of PBPs. It has a central core which is made of 2-5 allophycocyanin 19

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(APC) cylinders, surrounded by 6-8 rods7,17 (Figure 1a). All rods contain phycocyanin (PC) and 20 2

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occasionally, other hypsochromic variants of PBPs as well. This mega-structure architecture 24

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contains additional non-pigment binding proteins called linker proteins (Lp).18 These Lp have a 26

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critical role in determining the directionality of EET7. 27 29

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PBPs are assembled from two types of subunits that form the (αβ) monomer. The PC monomer 31

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has three phycocyanobilin (PCB) chromophores: β155, α84 and β8419–21 (Figure 1b) while APC 32 3

contains only two PCBs: α84 and β8422–25 (Figure 1c). Monomers further assemble into trimers, 36

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hexamers and finally into rod or core cylinders. These further assemble to form the complete 37 38

PBS. Although PC and APC chromophores are chemically identical, their excited state energy 39 40

levels are different, due to the influence of the surrounding protein.26 43

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Photons are absorbed by PBP chromophores, creating an exciton. This exciton was suggested to 4 45

hop from one chromophore to another by Förster Resonance Energy Transfer (FRET) or by 46 48

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stronger excitonic quantum coupling.27–31 It migrates within and along the PC rods to the APC 50

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central core, finally reaching its terminal emitter and then to the reaction centers (RC).6 51 52 53 54 5 56 57 58 59 60 ACS Paragon Plus Environment

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The Journal of Physical Chemistry

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Figure 1. Cyanobacterial photosynthetic antenna proteins structure. (a) Schematic representation 18

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of typical tri-cylindrical phycobilisome light harvesting antenna, composed of an APC 19 21

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containing a core (dark blue) and PC rods surrounding the core (cyan). (b) Crystallographic 23

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derived phycocyanin hexamer structure contains 18 chromophores of three kinds: β155 (blue), 25

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α84 (cyan) and β84 (red). PDB ID: 3O18.21 (c) Allophycocyanin hexamer contains 12 26 27

chromophores of two kinds: α84 (cyan) and β84 (red). PDB ID: 3DBJ.24 29

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The PBS super-structure is highly adaptive to changing environmental conditions.7,32–34 34 35

Although extensive efforts have been made to determine its ultra-structure by X-ray 36 37

crystallography or electron-microscopy, so far only partial successes have been reported7 and it 40

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is now clear that structural dynamics are of functional importance. 41 42

One example of a structural dynamic has been identified in desert sand crust cyanobacteria. This 43 45

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species can tolerate almost complete desiccation and sustain both hot and arid conditions by 47

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switching from a wet phase to a desiccated phase. In the latter phase, conformational changes to 48 49

its PBSs were shown to occur and to be related to photosynthesis quenching mechanisms.35 50 52

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In this research, we explore the optical properties of PC and APC isolated from the thermophilic 54

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cyanobacterium Thermosynechococcus vulcanus in two phases: wet and desiccated, to elucidate 5 57

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their EET mechanisms. We present both experimental evidence and theoretical calculations for 58 59 60 ACS Paragon Plus Environment

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two modes of operation: One that allows energy funneling towards the APC core, and another 5

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that induces energy quenching, utilizing the same photosynthetic chromophore. 6 7 8 10

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EXPERIMENTAL METHODS 1 12

The experimental procedure consists of isolation of PC and APC proteins, followed by re13 15

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suspension in six different buffer solutions (Table S1) and measurement of their optical 17

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properties in their completely wet phase and in the desiccated phase. 18 19

Protein preparation 20 2

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T. vulcanus cells were grown in a 2-liter temperature-controlled growth chamber in BG11 24

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medium supplemented with 5% CO2 in air at 55 C, with fluorescent lamp illumination. Cells 25 27

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were grown for 5 to 7 days before collection by centrifugation (6,000 × g for 15 min). The cells 29

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were re-suspended in isolation buffer (20 mM Tris, 10 mM MgCl2 and 10 mM CaCl2, pH 7.5) 31

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and treated with lysozyme (1 mg/ml) for 2 h at 50 C in the dark, before passing through a 32 34

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microfluidizer (M-110S) at maximal pressure. The sample was then centrifuged for 15 min at 36

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1,200 × g to discard the unbroken cell fraction. The green supernatant was then incubated with 37 38

3% (vol/vol) Triton-X for 60 min at room temperature and centrifuged for 45 min at 27,500 × g. 39 41

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The supernatant containing the soluble PBP was separated using low pressure anion exchange 43

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chromatography (Toyopearl QAE-550C filled as resin). Fractions were characterized by 4 45

absorption spectroscopy in order to select samples containing pure PBP. APC was obtained in 46 48

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high purity after this step. PC was dialyzed and further purified by high-pressure anion exchange 50

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column (Agilent PL-SAX 1000 Å 8 µ column). The resulting pure PC and APC were exchanged 51 53

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into 10 mM Potassium Phosphate (K-Pho), pH=7.5 using ultrafiltration (Millipore). The total 5

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volume of each sample was divided equally to yield 6 x 400 µl of PC at 10 mg/ml and 6 x 400 µl 56 57 58 59 60 ACS Paragon Plus Environment

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The Journal of Physical Chemistry

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of APC at 3 mg/ml. Additional buffer exchanges were done in similar fashion for each sample, 5

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generating 6 samples of PC and 6 samples of APC containing different buffers (Table S1). One 6 8

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sample was dialyzed against DDW to achieve a very low salt concentration (25 µM). This 10

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sample is attributed as low salts (LS). Two other samples were dialyzed against 10 mM Tris 1 12

HCL and Tris HBr buffers. The last three samples were dialyzed against 10 mM, 500 mM and 13 15

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900 mM potassium phosphate buffer. In this article, all figures show data for LS samples except 17

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for Figure 3 and Figures S1-S4. 18 19

Optical Measurements 20 2

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Absorption measurements were performed using an integrating sphere. A white light source 24

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(ThorLabs QTH10) was coupled into a 150 mm diameter integrating sphere via a lens. The 25 27

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samples were then placed over the exit aperture. In the experimental setup the aperture was 29

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bigger than the sample. In this way the sample was equally illuminated from all directions by the 31

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integrating sphere. The transmitted light was collected by a lens coupled to an optic fiber that 32 34

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was connected to a visible range fiber spectrometer (OceanOptics USB4000). Spectrometer 36

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measurement error is