Excitation energy transfer between Light-harvesting complex II and Photosystem I in reconstituted membranes.

Light-harvesting complex II (LHCII), the major peripheral antenna of Photosystem II in plants, participates in several concerted mechanisms for regulation of the excitation energy and electron fluxes in thylakoid membranes. In part, these include interaction of LHCII with Photosystem I (PSI) enhancing the latter's absorption cross-section - for example in the well-known state 1 - state 2 transitions or as a long-term acclimation to high light. In this work we examined the capability of LHCII to deliver excitations to PSI in reconstituted membranes in vitro. Proteoliposomes with native plant thylakoid membrane lipids and different stoichiometric ratios of LHCII:PSI were reconstituted and studied by steady-state and time-resolved fluorescence spectroscopy. Fluorescence emission from LHCII was strongly decreased in PSI-LHCII membranes due to trapping of excitations by PSI. Kinetic modelling of the time-resolved fluorescence data revealed the existence of separate pools of LHCII distinguished by the time scale of energy transfer. A strongly coupled pool, equivalent to one LHCII trimer per PSI, transferred excitations to PSI with near-unity efficiency on a time scale of less than 10ps but extra LHCIIs also contributed significantly to the effective antenna size of PSI, which could be increased by up to 47% in membranes containing 3 LHCII trimers per PSI. The results demonstrate a remarkable competence of LHCII to increase the absorption cross-section of PSI, given the opportunity that the two types of complexes interact in the membrane.

Pigment interactions in light-harvesting complex II in different molecular environments.

Extraction of plant light-harvesting complex II (LHCII) from the native thylakoid membrane or from aggregates by the use of surfactants brings about significant changes in the excitonic circular dichroism (CD) spectrum and fluorescence quantum yield. To elucidate the cause of these changes, e.g. trimer-trimer contacts or surfactant-induced structural perturbations, we compared the CD spectra and fluorescence kinetics of LHCII aggregates, artificial and native LHCII-lipid membranes, and LHCII solubilized in different detergents or trapped in polymer gel. By this means we were able to identify CD spectral changes specific to LHCII-LHCII interactions, at (-)-437 and (+)-484 nm, and changes specific to the interaction with the detergent n-dodecyl-ß-maltoside (ß-DM) or membrane lipids, at (+)-447 and (-)-494 nm. The latter change is attributed to the conformational change of the LHCII-bound carotenoid neoxanthin, by analyzing the CD spectra of neoxanthin-deficient plant thylakoid membranes. The neoxanthin-specific band at (-)-494 nm was not pronounced in LHCII in detergent-free gels or solubilized in the α isomer of DM but was present when LHCII was reconstituted in membranes composed of phosphatidylcholine or plant thylakoid lipids, indicating that the conformation of neoxanthin is sensitive to the molecular environment. Neither the aggregation-specific CD bands, nor the surfactant-specific bands were positively associated with the onset of fluorescence quenching, which could be triggered without invoking such spectral changes. Significant quenching was not active in reconstituted LHCII proteoliposomes, whereas a high degree of energetic connectivity, depending on the lipid:protein ratio, in these membranes allows for efficient light harvesting.

Preparation, purification, and characterization of aminopropyl-functionalized silica sol.

A new, simple, and "green" method was developed for the surface modification of 20 nm diameter Stöber silica particles with 3-aminopropyl(diethoxy)methylsilane in ethanol. The bulk polycondensation of the reagent was inhibited and the stability of the sol preserved by adding a small amount of glacial acetic acid after appropriate reaction time. Centrifugation, ultrafiltration, and dialysis were compared in order to choose a convenient purification technique that allows the separation of unreacted silylating agent from the nanoparticles without destabilizing the sol. The exchange of the solvent to acidic water during the purification yielded a stable colloid, as well. Structural and morphological analysis of the obtained aminopropyl silica was performed using transmission electron microscopy (TEM), dynamic light scattering (DLS) and zeta potential measurements, Fourier-transform infrared (FTIR), (13)C and (29)Si MAS nuclear magnetic resonance (NMR) spectroscopies, as well as small angle X-ray scattering (SAXS). Our investigations revealed that the silica nanoparticle surfaces were partially covered with aminopropyl groups, and multilayer adsorption followed by polycondensation of the silylating reagent was successfully avoided. The resulting stable aminopropyl silica sol (ethanolic or aqueous) is suitable for biomedical uses due to its purity.