Fluorescence Microscopy of Single Liposomes with Incorporated Pigment-Proteins

Reconstitution of transmembrane proteins into liposomes is a widely used method to study their behavior under conditions closely resembling the natural ones. However, this approach does not allow precise control of the liposome size, reconstitution efficiency, and the actual protein-to-lipid ratio in the formed proteoliposomes, which might be critical for some applications and/or interpretation of data acquired during the spectroscopic measurements. Here, we present a novel strategy employing methods of proteoliposome preparation, fluorescent labeling, purification, and surface immobilization that allow us to quantify these properties using fluorescence microscopy at the singleliposome level and for the first time apply it to study photosynthetic pigment protein complexes LHCII. We show that LHCII proteoliposome samples, even after purification with a density gradient, always contain a fraction of nonreconstituted protein and are extremely heterogeneous in both protein density and liposome sizes. This strategy enables quantitative analysis of the reconstitution efficiency of different protocols and precise fluorescence spectroscopic study of various transmembrane proteins in a controlled nativelike environment

DataSheet_1_Energy transfer from phycobilisomes to photosystem I at room temperature.pdf

The phycobilisomes function as the primary light-harvesting antennae in cyanobacteria and red algae, effectively harvesting and transferring excitation energy to both photosystems. Here we investigate the direct energy transfer route from the phycobilisomes to photosystem I at room temperature in a mutant of the cyanobacterium Synechocystis sp. PCC 6803 that lacks photosystem II. The excitation dynamics are studied by picosecond time-resolved fluorescence measurements in combination with global and target analysis. Global analysis revealed several fast equilibration time scales and a decay of the equilibrated system with a time constant of ≈220 ps. From simultaneous target analysis of measurements with two different excitations of 400 nm (chlorophyll a) and 580 nm (phycobilisomes) a transfer rate of 42 ns-1 from the terminal emitter of the phycobilisome to photosystem I was estimated.</p

Two-Dimensional Spectroscopy of Chlorophyll <i>a</i> Excited-State Equilibration in Light-Harvesting Complex II

Excited-state relaxation dynamics and energy-transfer processes in the chlorophyll <i>a</i> (Chl <i>a</i>) manifold of the light-harvesting complex II (LHCII) were examined at physiological temperature using femtosecond two-dimensional electronic spectroscopy (2DES). The experiments were done under conditions free from singlet–singlet annihilation and anisotropic decay. Energy transfer between the different domains of the Chl <i>a</i> manifold was found to proceed on time scales from hundreds of femtoseconds to five picoseconds, before reaching equilibration. No component slower than 10 ps was observed in the spectral equilibration dynamics. We clearly observe the bidirectional (uphill and downhill) energy transfer of the equilibration process between excited states. This bidirectional energy flow, although implicit in the modeling and simulation of the EET processes, has not been observed in any prior transient absorption studies. Furthermore, we identified the spectral forms associated with the different energy transfer lifetimes in the equilibration process

Process Development of the Soft Histone Deacetylate Enzyme Inhibitor SHP-141: Acylation of Methyl Paraben and Suberyl Hydroxamic Acid Formation

SHP-141 (<b>1</b>) is a hydroxamic acid-based inhibitor of histone deacetylase enzymes which is under development for the treatment of cutaneous T-cell lymphoma. The original synthesis of <b>1</b> involved five synthetic steps beginning with suberic acid monomethyl ester. Final deprotection of the <i>O</i>-benzyl hydroxamate moiety using hydrogen and palladium catalyst mandated the use of metal scavengers to reduce palladium levels to within International Council for Harmonisation (ICH) guidance. Owing to the sensitivity of <b>1</b> toward self-condensation and the potential for N–O bond cleavage under hydrogenolytic conditions, we developed an alternative route to <b>1</b> which avoids Pd-mediated hydrogenation and prolonged metal scavenger treatment. This two-step process employs readily available suberic acid and methyl paraben and has successfully delivered multiple kilograms of <b>1</b> for clinical use. Importantly, crude <b>1</b> was stabilized for recrystallization in acetonitrile (ACN) solution by the addition of 0.1% citric acid and 4% water. Additionally, the filtration and drying of suitably sized aggregates of <b>1</b> with high purity (100 area%) was accomplished via temperature cycling of the <b>1</b>/ACN solution