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A New Group of Eubacterial Light-Driven Proton Pumps Lacking the Carboxylic Proton Donor
Monday, February 29, 2016 mechanism has advantages over other, arguably simpler, mechanisms. For example, ATP-hydrolyzing P-loop transporters can be reversed to perform ATP synthesis, suggesting a variety of potential synthesis mechanisms. The present work uses kinetic models, operating under fundamental biophysical constraints, to compare a rotary-like mechanism for ATP synthesis with a systematic enumeration of alternative mechanisms based on event-ordering; each is constrained to operate under the same Hþ/ATP ratio and thermodynamic conditions. The models are independent of structural details and rely solely on thermodynamic and kinetic principles. The resulting models qualitatively reproduce key kinetic characteristics of ATP synthase under physiological conditions, including the sigmoidal relationship between the rate of ATP synthesis and pmf. When the mechanisms are separately optimized to maximize the rate of ATP synthesis over a range of presumed physiological conditions, the performance of the rotary mechanism stands out significantly, particularly in the most challenging, energy-poor conditions. Although all the models are thermodynamically equivalent in using the same free energy per ATP synthesized, the rotary model possesses a kinetic advantage: its one-at-a-time transport of protons enables more ratchet-like progress and less delay-induced unbinding of protons on the driving (low-pH) side of the membrane. Our results suggest the rotary mechanism has a quantifiable intrinsic advantage that may have played a role in evolution. 1535-Pos Board B512 A New Group of Eubacterial Light-Driven Proton Pumps Lacking the Carboxylic Proton Donor Andrew Harris1, Milena Ljumovic1, Ana-Nicoleta Bondar2, Yohei Shibata3, Yuto Suzuki3, Shota Ito3, Keiichi Inoue3, Hideki Kandori3, Leonid Brown1. 1 Physics, University of Guelph, Guelph, ON, Canada, 2Physics, Freie University, Berlin, Germany, 3Frontier Materials, Nagoya Institute of Technology, Nagoya, Japan. One of the main functions of microbial rhodopsins is outward-directed lightdriven proton transport across the plasma membrane, which can provide sources of energy alternative to respiration and chlorophyll photosynthesis. Proton-pumping rhodopsins are found in Archaea (Halobacteria), multiple groups of Bacteria, numerous fungi, and some microscopic algae. An overwhelming majority of these proton pumps share the common transport mechanism, in which a proton from the retinal Schiff base is first transferred to the primary proton acceptor (normally an Asp) on the extracellular side of retinal. Next, reprotonation of the Schiff base from the cytoplasmic side is mediated by a carboxylic proton donor (Asp or Glu), which is located on helix C and is usually hydrogen-bonded to Thr or Ser on helix B. The only notable exception from this trend was recently found in Exiguobacterium, where the carboxylic proton donor is replaced by Lys. Here we describe a new group of efficient proteobacterial retinal-binding light-driven proton pumps which lack the carboxylic proton donor on helix C (most often replaced by Gly) but possess a unique His residue on helix B. We characterize the group spectroscopically and propose that this histidine forms a proton-donating complex compensating for the loss of the carboxylic proton donor. 1536-Pos Board B513 Photo-Current and TEM Imaging Characterization of Light-Gated Ion Pump Proteins in Lipid Membranes Joel Kamwa, Surendra Singh, Jiali Li. University of Arkansas, Fayetteville, AR, USA. We report our study on TEM imaging, and photocurrent characterization of two photosynthetic proteins, Bacteriorhodopsin (BR), and Halorhodopsin (HR), which act as light-gated ion pumps. Bacteriorhodopsin acts as a proton (Hþ) pump, and Halorhodopsin as a chloride ion (Cl-) pump. Both proteins are reconstituted in nano-bilayer membranes supported by Teflon and silicon-based substrates. When a beam of green or blue light shines on the membranes, it induces ion (Hþ or Cl-) transport, which is measurable as a photocurrent. We measure how the photocurrent generated by BR and HR changes as a function of their concentration in the membranes and cross-membrane potential. Furthermore, we take TEM images of the membranes with BR and HR reconstituted, to characterize the structure and the density of the proteins in the lipid membranes. The relationship between these structures and the generated photocurrent will also be discussed. 1537-Pos Board B514 A DNA-Based Building Block for Designer Excitonic Circuits Etienne Boulais1, Nicolas Sawaya2, Re´mi Veneziano1, Alessio Andreoni3, Su Lin3, Neal Woodbury3, Hao Yan3, Alan Aspuru-Guzik2, Mark Bathe1. 1 Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA, 2Chemistry and Chemical Biology, Harvard University,
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Cambridge, MA, USA, 3Chemistry and Biochemistry, Arizona State University, Tempe, AZ, USA. Mimicking the ability of natural light harvesting systems to absorb and transport energy efficiently over long ranges is an important unmet challenge in bionanotechnology. Structured biomolecular scaffolds such as DNA assemblies offer exquisite control over 3D nanometer-scale chromophore organization to program custom energy transfer pathways at the nanoscale while exploiting the quantum nature of inter-molecular chromophore coupling. Here, we report a bottom-up multi-chromophore design framework that leverages quantum properties to design modular, customizable excitonic circuits using programmed DNA assemblies. To achieve this we employ sequence-specific DNA scaffolds that organize strongly interacting cyanine aggregates as building blocks to construct fast, coherent energy transfer in complex supramolecular architectures. Using this approach, we synthesized a DX-tile that consists of multiple interacting cyanine aggregate units to attain spectral tunability, superradiance and fast exciton transport. Because of the increasing number of synthetic chromophores available and the structural flexibility and control of programmable DNA nanotechnology, our bottom-up approach may enable rational design of a broad array of nanoscale excitonic circuits in large-scale one-, two-, and three-dimensional DNA-based arrays. 1538-Pos Board B515 Photoinduced Electron Transfer from Porphyrins to Quinones Randomly Dispersed in a Polymeric Medium Marcelo K.K. Nakaema1, Rosemary Sanches2. 1 Escola de Cieˆncias e Tecnologia, Universidade Federal do Rio Grande do Norte, Natal, Brazil, 2Instituto de Fı´sica de Sa˜o Carlos, Universidade de Sa˜o Paulo, Sa˜o Carlos, Brazil. The electron transfer rate from porphyrins to quinones molecules randomly dispersed in a polymeric medium was investigated by time-resolved fluorescence spectroscopy. The rate constant was supposed to vary exponentially with the distance separating the donor and the acceptor molecules [k = Z*exp(-alpha*r)]. The results were discussed in terms of the random distribution of acceptors model (Inokuti-Hirayama model). For the pair octaethylporphyrin (OEP)/duroquinone (DQ) the estimated pre-exponential factor in the rate constant (Z) was 910 s-1 and the distance decay coefficient (alpha) was ˚ -1. For the other two pairs, OEP/tetrachlorobenzoquinone (TCQ) and tet0.76 A raphenylporphyrin (TPP)/DQ only the ratio Z/alpha3 was determined. 1539-Pos Board B516 The Role of Protein Conformational Changes in Tuning the Fluorescence State of Light-Harvesting Complexes Nicoletta Liguori1, Xavier Periole2, Laura M. Roy1, Yarah Bot1, Siewert J. Marrink2, Roberta Croce1. 1 Department of Physics and Astronomy and Institute for Lasers, Life and Biophotonics, VU University of Amsterdam, Amsterdam, Netherlands, 2 Faculty of Mathematics and Natural Sciences, University of Groningen, Groningen, Netherlands. In a world undergoing rapid changes in sunlight quality and quantity, the successful design of photosynthesis must rely on the ability of plants and algae to quickly and reversibly adapt to the different conditions. A fast response to the fluctuating environment is given by the Light-Harvesting Complexes (LHCs). LHCs are large pigment-protein complexes responsible for the first steps of photosynthesis. LHCs capture light and deliver excitations to Photosystem I and II (PSI and PSII) where photosynthesis starts. Spectroscopy studies showed that LHCs can switch between states of long and short fluorescence lifetime. Quenched fluorescence states help preventing reactions with oxygen eventually leading to oxidative stress. Although postulated for long time, the role of protein conformational changes in tuning optical properties of LHCs remained unconfirmed until only recently. We here present the role of specific protein domains in tuning pigment-pigment interactions in a series of LHCs. A first example is LHCSR3, the trigger of photoprotection in the green alga Chlamydomonas reinhardtii, which possesses a peculiar C-terminus. This domain is pH-sensitive (pH is a stress-indicator in the alga) and causes tuning of fluorescence to quenched states upon acidification [Liguori, N. et al JACS 2013]. Additionally we show that this mechanism is absent in all related LHC proteins from PSII, which indeed do not present the same residue composition at this domain [Liguori, N. et al in preparation]. However LHCII, which is the most abundant LHC in both plants and algae, still undergoes significant conformational changes at a different domain: the N-terminus. It is a highly disordered domain and, as we show, a strong correlation is found between the different conformations of this domain and the different pigment-pigment interactions [Liguori, N. et al Scientific Reports in press].
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Cambridge, MA, USA, 3Chemistry and Biochemistry, Arizona State University, Tempe, AZ, USA. Mimicking the ability of natural light harvesting systems to absorb and transport energy efficiently over long ranges is an important unmet challenge in bionanotechnology. Structured biomolecular scaffolds such as DNA assemblies offer exquisite control over 3D nanometer-scale chromophore organization to program custom energy transfer pathways at the nanoscale while exploiting the quantum nature of inter-molecular chromophore coupling. Here, we report a bottom-up multi-chromophore design framework that leverages quantum properties to design modular, customizable excitonic circuits using programmed DNA assemblies. To achieve this we employ sequence-specific DNA scaffolds that organize strongly interacting cyanine aggregates as building blocks to construct fast, coherent energy transfer in complex supramolecular architectures. Using this approach, we synthesized a DX-tile that consists of multiple interacting cyanine aggregate units to attain spectral tunability, superradiance and fast exciton transport. Because of the increasing number of synthetic chromophores available and the structural flexibility and control of programmable DNA nanotechnology, our bottom-up approach may enable rational design of a broad array of nanoscale excitonic circuits in large-scale one-, two-, and three-dimensional DNA-based arrays. 1538-Pos Board B515 Photoinduced Electron Transfer from Porphyrins to Quinones Randomly Dispersed in a Polymeric Medium Marcelo K.K. Nakaema1, Rosemary Sanches2. 1 Escola de Cieˆncias e Tecnologia, Universidade Federal do Rio Grande do Norte, Natal, Brazil, 2Instituto de Fı´sica de Sa˜o Carlos, Universidade de Sa˜o Paulo, Sa˜o Carlos, Brazil. The electron transfer rate from porphyrins to quinones molecules randomly dispersed in a polymeric medium was investigated by time-resolved fluorescence spectroscopy. The rate constant was supposed to vary exponentially with the distance separating the donor and the acceptor molecules [k = Z*exp(-alpha*r)]. The results were discussed in terms of the random distribution of acceptors model (Inokuti-Hirayama model). For the pair octaethylporphyrin (OEP)/duroquinone (DQ) the estimated pre-exponential factor in the rate constant (Z) was 910 s-1 and the distance decay coefficient (alpha) was ˚ -1. For the other two pairs, OEP/tetrachlorobenzoquinone (TCQ) and tet0.76 A raphenylporphyrin (TPP)/DQ only the ratio Z/alpha3 was determined. 1539-Pos Board B516 The Role of Protein Conformational Changes in Tuning the Fluorescence State of Light-Harvesting Complexes Nicoletta Liguori1, Xavier Periole2, Laura M. Roy1, Yarah Bot1, Siewert J. Marrink2, Roberta Croce1. 1 Department of Physics and Astronomy and Institute for Lasers, Life and Biophotonics, VU University of Amsterdam, Amsterdam, Netherlands, 2 Faculty of Mathematics and Natural Sciences, University of Groningen, Groningen, Netherlands. In a world undergoing rapid changes in sunlight quality and quantity, the successful design of photosynthesis must rely on the ability of plants and algae to quickly and reversibly adapt to the different conditions. A fast response to the fluctuating environment is given by the Light-Harvesting Complexes (LHCs). LHCs are large pigment-protein complexes responsible for the first steps of photosynthesis. LHCs capture light and deliver excitations to Photosystem I and II (PSI and PSII) where photosynthesis starts. Spectroscopy studies showed that LHCs can switch between states of long and short fluorescence lifetime. Quenched fluorescence states help preventing reactions with oxygen eventually leading to oxidative stress. Although postulated for long time, the role of protein conformational changes in tuning optical properties of LHCs remained unconfirmed until only recently. We here present the role of specific protein domains in tuning pigment-pigment interactions in a series of LHCs. A first example is LHCSR3, the trigger of photoprotection in the green alga Chlamydomonas reinhardtii, which possesses a peculiar C-terminus. This domain is pH-sensitive (pH is a stress-indicator in the alga) and causes tuning of fluorescence to quenched states upon acidification [Liguori, N. et al JACS 2013]. Additionally we show that this mechanism is absent in all related LHC proteins from PSII, which indeed do not present the same residue composition at this domain [Liguori, N. et al in preparation]. However LHCII, which is the most abundant LHC in both plants and algae, still undergoes significant conformational changes at a different domain: the N-terminus. It is a highly disordered domain and, as we show, a strong correlation is found between the different conformations of this domain and the different pigment-pigment interactions [Liguori, N. et al Scientific Reports in press].