8 research outputs found
Biologischer Lichtsammler (LHCII) fĂŒr Halbleiternanokristalle (Quantum Dots)
Der light harvesting complex II (LHCII) ist ein pflanzliches Membranprotein, das in seiner trimeren Form ĂŒber 40 Chlorophylle bindet. In der Pflanze kann er besonders effizient Licht sammeln und die Anregungsenergie anschlieĂend fast verlustfrei ĂŒber andere chlorophyll-bindende Proteine an die Reaktionszentren weiterleiten. Aufgrund dieser besonderen Eigenschaften war es ein Ziel dieser Arbeit, rekombinanten LHCII mit synthetischen Komponenten zu kombinieren, die zur Ladungstrennung befĂ€higt sind. Zu diesem Zweck wurden unter anderem Halbleiternanokristalle (Quantum Dots, QDs) ausgewĂ€hlt, die je nach Zusammensetzung sowohl als Energieakzeptoren als auch als Energiedonoren in Frage kamen. Durch Optimierung des Puffers gelang es, die Fluoreszenzquantenausbeute der QDs in wĂ€ssriger Lösung zu erhöhen und zu stabilisieren, so dass die Grundvoraussetzungen fĂŒr die
spektroskopische Untersuchung verschiedener LHCII-QD-Hybridkomplexe erfĂŒllt waren.rnUnter Verwendung bereits etablierter AffinitĂ€tssequenzen zur Bindung des LHCII an die QDs konnte gezeigt werden, dass die in dieser Arbeit verwendeten Typ-I QDs aus CdSe und ZnS sich kaum als Energie-Donoren fĂŒr den LHCII eignen. Ein Hauptgrund lag im vergleichsweise kleinen Försterradius R0 von 4,1 nm. Im Gegensatz dazu wurde ein R0 von 6,4 nm fĂŒr den LHCII als Donor und Typ-II QDs aus CdTe, CdSe und ZnS als Akzeptor errechnet, wodurch in diesem System eine höhere Effizienz des Energietransfers zu erwarten war. Fluoreszenzspektroskopische Untersuchungen von Hybridkomplexen aus LHCII und Typ-II QDs ergaben eine hohe PlausibilitĂ€t fĂŒr einen Fluoreszenz Resonanz Energietransfer (FRET) vom Lichtsammler auf die QDs. Weitere QD-AffinitĂ€tssequenzen fĂŒr den LHCII wurden identifiziert und deren Bindekonstanten ermittelt. Versuche mit dem Elektronenakzeptor Methylviologen lieferten gute Hinweise auf eine LHCII-sensibilisierte
Ladungstrennung der Typ-II QDs, auch wenn dies noch anhand alternativer Messmethoden wie z.B. durch transiente Absorptionsspektroskopie bestĂ€tigt werden muss. rnEin weiteres Ziel war die Verwendung von LHCII als Lichtsammler in dye-sensitized solar cells (DSSC). Geeignete dotierte TiO2-Platten wurden ermittelt, das Verfahren zur Belegung der Platten optimiert und daher mit wenig Aufwand eine hohe LHCII-Belegungsdichte erzielt. Erste Messungen von Aktionsspektren mit LHCII und einem zur Ladungstrennung fĂ€higen Rylenfarbstoff zeigen eine, wenn auch geringe, LHCII sensibilisierte Ladungstrennung. rnDie Verwendung von Lanthanide-Binding-Tags (LBTs) ist ein potentielles Verfahren zur in vivo-Markierung von Proteinen mit Lanthanoiden wie Europium und Terbium. Diese Metalle besitzen eine ĂŒberdurchschnittlich lange Lumineszenzlebensdauer, so dass sie leicht von anderen fluoreszierenden MolekĂŒlen unterschieden werden können. Im Rahmen der vorliegenden Arbeit gelang es, eine LBT in rekombinanten LHCII einzubauen und
einen Lumineszenz Resonanz Energietransfer (LRET) vom Europium auf den LHCII nachzuweisen.rnThe light harvesting complex II (LHCII) is a membrane protein and consists of more than 40 chlorophylls in its trimeric version. In plants it performs efficient light harvesting and transfers the excitation energy nearly quantitatively via other pigment-binding proteins to the reaction center. Due to these LHCII properties it was of interest to combine recombinant LHCII with synthetic compounds that are capable of charge separation. To this end semiconductor nanocrystals, so-called Quantum dots (QDs), where chosen as energy acceptors. Depending on their composition, QDs can also serve as energy donors. By optimizing the buffer system, the QDs fluorescence quantum yield in
aqueous solution has been enhanced and stabilized, fulfilling the prerequisites for spectroscopic investigations of different LHCII-QD hybrid complexes.rnBy using established affinity tags to bind LHCII to QDs it was shown that type-I nanocrystals from CdSe and ZnS were no efficient energy donors for LHCII, presumably due to the small Förster radius (R0) of 4.1 nm. By contrast, a larger R0 of 6.4 nm was estimated for hybrid complexes of LHCII as donors and type-II QDs (CdTe, CdSe, ZnS), thus a higher efficiency of energy transfer was expected. Complexes of LHCII and type-II QDs exhibited fluorescence properties that were indicative of Foerster-type energy transfer from LHCII to QD. Additional QD-affinity tags have been established for the LHCII and their binding constants were estimated. Experiments with the electron acceptor methyl viologen indicated an LHCII sensitized charge separation in QDs. This preliminary result still needs to be confirmed, for example transient absorption spectroscopy.rnAnother
objective was to integrate LHCII-hybrid complexes into dye-sensitized solar cells (DSSCs). Suitably doted TiO2 plates were loaded by an optimized procedure, enhancing the LHCII density on the plates. Preliminary recordings of action spectra with LHCII and a rylen dye as a sensitizer showed a small but significant LHCII-sensitized charge separation. rnThe use of lanthanide binding tags (LBTs) is a possibility for in vivo labeling of proteins with lanthanides like terbium and europium. These metals have an extraordinary long luminescence lifetime making them easily distinguishable from other fluorescent molecules. In this work an LBT was introduced into recombinant LHCII and luminescence resonance energy transfer was shown to take place from europium to LHCII.r
Bio serves nano : biological light-harvesting complex as energy donor for Semiconductor quantum dots
Stability of water-soluble chlorophyll protein (WSCP) depends on phytyl conformation
Water-soluble chlorophyll proteins (WSCP) from Brassicaceae form homotetrameric chlorophyll (Chl)âprotein complexes binding one Chl per apoprotein and no carotenoids. Despite the lack of photoprotecting photoprotecting pigments, the complex-bound Chls displays a remarkable stability toward photodynamic photodynamic damage. On the basis of a mutational study, we show that not only the presence of the phytyls phytyls is necessary for photoprotection in WSCPs, as we previously demonstrated, but also is their correct correct conformation and localization. The extreme heat stability of WSCP also depends on the presence presence of the phytyl chains, confirming their relevance for the unusual stability of WSCP
Water-Soluble Chlorophyll Protein (WSCP) Stably Binds Two or Four Chlorophylls
Water-soluble chlorophyll
proteins (WSCPs) of class IIa from Brassicaceae
form tetrameric complexes containing one chlorophyll (Chl) per apoprotein
but no carotenoids. The complexes are remarkably stable toward dissociation
and protein denaturation even at 100 °C and extreme pH values,
and the Chls are partially protected against photooxidation. There
are several hypotheses that explain the biological role of WSCPs,
one of them proposing that they function as a scavenger of Chls set
free upon plant senescence or pathogen attack. The biochemical properties
of WSCP described in this paper are consistent with the protein acting
as an efficient and flexible Chl scavenger. At limiting Chl concentrations,
the recombinant WSCP apoprotein binds substoichiometric amounts of
Chl (two Chls per tetramer) to form complexes that are as stable toward
thermal dissociation, denaturation, and photodamage as the fully pigmented
ones. If more Chl is added, these two-Chl complexes can bind another
two Chls to reach the fully pigmented state. The protection of WSCP
Chls against photodamage has been attributed to the apoprotein serving
as a diffusion barrier for oxygen, preventing its access to triplet
excited Chls and, thus, the formation of singlet oxygen. By contrast,
the sequential binding of Chls by WSCP suggests a partially open or
at least flexible structure, raising the question of how WSCP photoprotects
its Chls without the help of carotenoids
Bio Serves Nano: Biological Light-Harvesting Complex as Energy Donor for Semiconductor Quantum Dots
Light-harvesting complex (LHCII) of the photosynthetic
apparatus
in plants is attached to type-II coreâshell CdTe/CdSe/ZnS nanocrystals
(quantum dots, QD) exhibiting an absorption band at 710 nm and carrying
a dihydrolipoic acid coating for water solubility. LHCII stays functional
upon binding to the QD surface and enhances the light utilization
of the QDs significantly, similar to its light-harvesting function
in photosynthesis. Electronic excitation energy transfer of about
50% efficiency is shown by donor (LHCII) fluorescence quenching as
well as sensitized acceptor (QD) emission and corroborated by time-resolved
fluorescence measurements. The energy transfer efficiency is commensurable
with the expected efficiency calculated according to FoÌrster
theory on the basis of the estimated donorâacceptor separation.
Light harvesting is particularly efficient in the red spectral domain
where QD absorption is relatively low. Excitation over the entire
visible spectrum is further improved by complementing the biological
pigments in LHCII with a dye attached to the apoprotein; the dye has
been chosen to absorb in the âgreen gapâ of the LHCII
absorption spectrum and transfers its excitation energy ultimately
to QD. This is the first report of a biological light-harvesting complex
serving an inorganic semiconductor nanocrystal. Due to the charge
separation between the core and the shell in type-II QDs the presented
LHCIIâQD hybrid complexes are potentially interesting for sensitized
charge-transfer and photovoltaic applications
Chlorophyll a/b binding-specificity in water-soluble chlorophyll protein
We altered the chlorophyll (Chl) binding sites in various versions of water-soluble chlorophyll protein (WSCP) by amino acid exchanges to alter their preferences for either Chl a or Chl b. WSCP is ideally suited for this mutational analysis since it forms a tetrameric complex with only four identical Chl binding sites. A loop of 4â6 amino acids is responsible for Chl a versus Chl b selectivity. We show that a single amino acid exchange within this loop changes the relative Chl a/b affinities by a factor of 40. We obtained crystal structures of this WSCP variant binding either Chl a or Chl b. The Chl binding sites in these structures were compared with those in the major light-harvesting complex (LHCII) of the photosynthetic apparatus in plants to search for similar structural features involved in Chl a/b binding specificity