Molecular model of LH1 light-harvesting complex of the photosynthetic center of Thermochromatium tepidum bacteria

A three-dimensional all-atom structure of the LH1 light-harvesting complex of the photosynthetic center of the Thermochromatium tepidum bacteria was constructed with molecular modeling techniques.     

Stability of integral membrane proteins under high hydrostatic pressure: the LH2 and LH3 antenna pigment-protein complexes from photosynthetic bacteria

The bacteriochlorophyll a-containing LH2 and LH3 antenna complexes are the integral membrane proteins that catalyze the photosynthetic process in purple photosynthetic bacteria. The LH2 complex from Rhodobacter sphaeroides shows characteristic strong absorbance at 800 and 850 nm due to the pigment molecules confined in two separate areas of the protein. In the LH3 complex from Rhodopesudomonas acidophila the corresponding bands peak at 800 and 820 nm. Using the bacteriochlorophyll a cofactors as intrinsic probes to monitor local changes in the protein structure, we investigate spectral responses of the antenna complexes to very high hydrostatic pressures up to 2.5 GPa when embedded into natural membrane environment or extracted with detergent. We first demonstrate that high pressure does induce significant alterations to the tertiary structure of the proteins not only in proximity of the 800 nm-absorbing bacteriochlorophyll a molecules known previously (Gall, A.; et al. Biochemistry 2003, 42, 13019) but also of the 850 nm- and 820 nm-absorbing molecules, including breakage of the hydrogen bond they are involved in. The membrane-protected complexes appear more resilient to damaging effects of the compression compared with the complexes extracted into mixed detergent-buffer environment. Increased resistance of the isolated complexes is observed at high protein concentration resulting aggregation as well as when cosolvent (glycerol) is added into the solution. These stability variations correlate with ability of penetration of the surrounding polar solvent (water) into the hydrophobic protein interiors, being thus the principal reason of the pressure-induced denaturation of the proteins. Considerable variability of elastic properties of the isolated complexes was also observed, tentatively assigned to heterogeneous protein packing in detergent micelles. While a number of the isolated complexes release most of their bacteriochlorophyll a content under high pressure, quite some of them remain apparently intact. The pigmented photosynthetic antenna complexes thus constitute a suitable model system for studying in detail the stability of integral membrane proteins.     

Modeling of photosynthetic light-harvesting: From structure to function

In order to bridge the gap between the crystal structure of photosynthetic pigment-protein complexes and the data gathered in optical experiments, two essential problems need to be solved. On one hand, theories of optical spectra and excitation energy transfer have to be developed that take into account the pigment-pigment (excitonic) and the pigment-protein (exciton-vibrational) coupling on an equal footing. On the other hand, the parameters entering these theories need to be calculated from the structural data. Good agreement between simulations and experimental data then allows to draw conclusions on structure-function relationships of these complexes and to make predictions. In the development of theory, a delicate question is how to describe the interplay between the quantum dynamics of excitons and the dephasing of coherences by the coupling of excitons to protein vibrations. Quantum mechanic coherences are utilized for efficient light harvesting. In the reaction centers of purple bacteria an energy sink is created by a coherent coupling of exciton states to intermolecular charge transfer states. The dephasing of coherences can be monitored, e.g., by the temperature dependent shift of optical lines. In the Fenna-Matthews-Olson protein, which acts as an excitation energy wire between the outer chlorosome antenna and the reaction center complex, an energy funnel for efficient light-harvesting is formed by the pigment-protein coupling. The protein shifts the local transition energies of the pigments, the so-called site energies in a specific way, such that pigments facing the reaction center are redshifted with respect to those on the chlorosome side. In the light-harvesting complex of higher plants an excitation energy funnel is created by the use of two different types of chlorophyll (Chl) pigments, Chla and Chlb and by the pigment-protein coupling that creates an energy sink at Chla 610 located in the stromal layer at the periphery of the complex. The close contact between Chla and Chlb gives rise to ultrafast subpicosecond exciton transfer, whereas dynamic localization effects are inferred to lead to long ps relaxation times between the majority of Chla pigments.     

Water dynamics in silica nanopores: the self-intermediate scattering functions

The dynamics of water molecules confined in approximately cylindrical silica nanopores is investigated using molecular simulation. The model systems are pores of diameter varying between 20 and 40 ? containing water at room temperature and at full hydration, prepared using grand canonical Monte Carlo simulation. Water dynamics in these systems is studied via molecular dynamics simulation. The results of the basic characterization of these systems have been reported in A. A. Milischuk and B. M. Ladanyi [J. Chem. Phys. 135, 174709 (2011)]. The main focus of the present study is the self-intermediate scattering function (ISF), F(S)(Q, t), of water hydrogens, the observable in quasi-elastic neutron scattering experiments. We investigate how F(S)(Q, t) depends on the pore diameter, the direction and magnitude of the momentum transfer Q, and the proximity of water molecules to the silica surface. We also study the contributions to F(S)(Q, t) from rotational and translational motions of water molecules and the extent of rotation-translation coupling present in F(S)(Q, t). We find that F(S)(Q, t) depends strongly on the pore diameter and that this dependence is due mainly to the contributions to the ISF from water translational motion and can be attributed to the decreased mobility of water molecules near the silica surface. The relaxation rate depends on the direction of Q and is faster for Q in the axial than in the radial direction. As the magnitude of Q increases, this difference diminishes but does not disappear. We find that its source is mainly the anisotropy in translational diffusion at low Q and in molecular reorientation at higher Q values.     

Ultrafast excitation relaxation in light-harvesting complex LH2 from Rb.sphaeroides 601

The light-driven reactions of photosynthesis are the means by which nature converts solar energy into electrochemical potential, which is eventually stored as chemical energy. These initial reactions occur in two closely coupled pigment systems, the network of so-called antenna system in which the excitation en-ergy is absorbed by the pigments and efficiently transported to another system, the photosynthetic reac-tion center where the energy is converted into a stable trans-membrane charge sepa…     

Directed formation of micro- and nanoscale patterns of functional light-harvesting LH2 complexes

The precision placement of the desired protein components on a suitable substrate is an essential prelude to any hybrid "biochip" device, but a second and equally important condition must also be met: the retention of full biological activity. Here we demonstrate the selective binding of an optically active membrane protein, the light-harvesting LH2 complex from Rhodobacter sphaeroides, to patterned self-assembled monolayers at the micron scale and the fabrication of nanometer-scale patterns of these molecules using near-field photolithographic methods. In contrast to plasma proteins, which are reversibly adsorbed on many surfaces, the LH2 complex is readily patterned simply by spatial control of surface polarity. Near-field photolithography has yielded rows of light-harvesting complexes only 98 nm wide. Retention of the native optical properties of patterned LH2 molecules was demonstrated using in situ fluorescence emission spectroscopy.     

Structure and dynamics of water confined in silica nanopores

We report the results of molecular simulation of water in silica nanopores at full hydration and room temperature. The model systems are approximately cylindrical pores in amorphous silica, with diameters ranging from 20 to 40 ?. The filled pores are prepared using grand canonical Monte Carlo simulation and molecular dynamics simulation is used to calculate the water structure and dynamics. We found that water forms two distinct molecular layers at the interface and exhibits uniform, but somewhat lower than bulk liquid, density in the core region. The hydrogen bond density profile follows similar trends, with lower than bulk density in the core and enhancements at the interface, due to hydrogen bonds between water and surface non-bridging oxygens and OH groups. Our studies of water dynamics included translational mean squared displacements, orientational time correlations, survival probabilities in interfacial shells, and hydrogen bond population relaxation. We found that the radial-axial anisotropy in translational motion largely follows the predictions of a model of free diffusion in a cylinder. However, both translational and rotational water mobilities are strongly dependent on the proximity to the interface, with pronounced slowdown in layers near the interface. Within these layers, the effects of interface curvature are relatively modest, with only a small increase in mobility in going from the 20 to 40 ? diameter pore. Hydrogen bond population relaxation is nearly bulk-like in the core, but considerably slower in the interfacial region.     

Melting and freezing of water in cylindrical silica nanopores

Freezing and melting of H(2)O and D(2)O in the cylindrical pores of well-characterized MCM-41 silica materials (pore diameters from 2.5 to 4.4 nm) was studied by differential scanning calorimetry (DSC) and (1)H NMR cryoporometry. Well-resolved DSC melting and freezing peaks were obtained for pore diameters down to 3.0 nm, but not in 2.5 nm pores. The pore size dependence of the melting point depression DeltaT(m) can be represented by the Gibbs-Thomson equation when the existence of a layer of nonfreezing water at the pore walls is taken into account. The DSC measurements also show that the hysteresis connected with the phase transition, and the melting enthalpy of water in the pores, both vanish near a pore diameter D* approximately equal to 2.8 nm. It is concluded that D* represents a lower limit for first-order melting/freezing in the pores. The NMR spin echo measurements show that a transition from low to high mobility of water molecules takes place in all MCM-41 materials, including the one with 2.5 nm pores, but the transition revealed by NMR occurs at a higher temperature than indicated by the DSC melting peaks. The disagreement between the NMR and DSC transition temperatures becomes more pronounced as the pore size decreases. This is attributed to the fact that with decreasing pore size an increasing fraction of the water molecules is situated in the first and second molecular layers next to the pore wall, and these molecules have slower dynamics than the molecules in the core of the pore.     

Carotenoid incorporation into microsomes: yields, stability and membrane dynamics

The carotenoids beta-carotene (BC), lycopene (LYC), lutein (LUT), zeaxanthin (ZEA), canthaxanthin (CTX) and astaxanthin (ASTA) have been incorporated into pig liver microsomes. Effective incorporation concentrations in the range of about 1-6 nmol/mg microsomal protein were obtained. A stability test at room temperature revealed that after 3 h BC and LYC had decayed totally whereas, gradually, CTX (46%), LUT (21%), ASTA (17%) and ZEA (5%) decayed. Biophysical parameters of the microsomal membrane were changed hardly by the incorporation of carotenoids. A small rigidification may occur. Membrane anisotropy seems to offer only a small tolerance for incorporation of carotenoids and seems to limit the achievable incorporation concentrations of the carotenoids into microsomes. Microsomes instead of liposomes should be preferred as a membrane model to study mutual effects of carotenoids and membrane dynamics.     

Microscopic visualization of alamethicin incorporation into model membrane monolayers

Lipid interactions and cooperative assembly properties are fundamental determinants for the action of antimicrobial membrane-active peptides. Here we analyze the interactions and aggregation properties of alamethicin, an antimicrobial pore-forming peptide, with films formed at the air/water interface. Surface-area/pressure isotherms, Brewster angle microscopy, and fluorescence-confocal microscopy provided detailed information on the morphologies and structural properties of the peptide and its effect on the film components. The pressure-area analysis and microscopy experiments facilitated unprecedented visualization of the structural consequences of alamethicin association at the air/water interface, with pure phospholipid films, and within mixed phospholipid/polydiacetylene (PDA) films. The analysis exposed the kinetic features and the interplay between the peptide aggregates and film constituents. In particular, the results demonstrate the use of phospholipid/PDA film assemblies for studying membrane-peptide association and interactions within two-dimensional films.     

Lipid composition-dependent incorporation of multiple membrane proteins into liposomes

Membrane proteins from bacteria Pasteurella multocida were used as a model for studying its incorporation into liposomes. An important step to achieve efficient high yield protein incorporation in proteoliposomes is the study of the more suitable lipid composition. To this end, we compared the amount of total protein, reconstituted by co-solubilization methods, into liposomes of phospholipids with different polar head groups and acyl chain lengths. The liposomes and proteoliposomes were characterised by isopycnic centrifugation in sucrose gradient and by dynamic light scattering. Experimental and theoretical results were compared considering the effects exerted through the hydrocarbon chain length, volume, and optimal cross-sectional area of the phospholipid (combined in the geometrical critical packing parameter, lipid–protein matching), critical spontaneous radius of curvature of the bilayer vesicle, phase transition temperature of the lipid and ratio of lipid–protein molecules present in the vesicles. The highest incorporation of multiple proteins was found with dipalmitoylphosphatidylcholine (DPPC), reaching a yield of 93% compared to the lower relative amounts incorporated in proteoliposomes of the other lipids. The incorporation of multiple proteins induces a proportional enhancement of vesicular dimension, since DPPC–proteoliposomes have an average diameter of 1850 Å, compared to the 1430 Å for pure DPPC vesicles.     

Optical transitions in the light-harvesting complexes of bacterial photosynthetic centers

The geometric parameters of the model structure of the bacterial photosynthetic center, which consists of a light-harvesting antenna complex, a reaction center, lipid layers, and the solvation shells of water molecules, were optimized using quantum mechanics-molecular mechanics (QM/MM). The optical absorption spectra of the bacteriochlorophyll were evaluated in the quantum subsystem. It was shown that the shift of the absorption bands towards longer wavelengths as compared to the spectrum of the individual bacteriochlorophyll molecules is characteristic of the cluster of pigment molecules that are found in the antennas.     

Structural characterization of surfactant aggregates adsorbed in cylindrical silica nanopores

The self-assembly of nonionic surfactants in the cylindrical pores of SBA-15 silica with a pore diameter of 8 nm was studied by small-angle neutron scattering (SANS) at different solvent contrasts. The alkyl ethoxylate surfactants C(10)E(5) and C(12)E(5) exhibit strong aggregative adsorption in the pores as indicated by the sigmoidal shape of the adsorption isotherms. The SANS intensity profiles can be represented by a sum of two terms, one accounting for diffuse scattering from surfactant aggregates in the pores and the other for Bragg scattering from the pore lattice of the silica matrix. The Bragg reflections are analyzed with a form factor model in which the radial density profile of the surfactant in the pore is approximated by a two-step function. Diffuse scattering is represented by a Teubner-Strey-type scattering function which indicates a preferred distance between adsorbed surface aggregates in the pores. Our results suggest that adsorption starts with formation of discrete surface aggregates which increase in number and eventually merge to interconnected patches as the plateau value of the adsorption isotherm is approached. A grossly different behavior, viz. formation of micelles as in solution, is found for the maltoside surfactant C(10)G(2), in agreement with the observed weak adsorption of this surfactant in SBA-15.     

Photosynthetic oxygen evolution in mesoporous silica material: adsorption of photosystem II reaction center complex into 23 nm nanopores in SBA

An oxygen-evolving photosynthetic reaction center complex (PSII) was adsorbed into nanopores in SBA, a mesoporous silica compound. We purified the dimer of PSII complex from a thermophilic cyanobacterium, Thermosynechococcus vulcanus, which grows optimally at 57 °C. The thermally stable PSII dimeric complex has a diameter of 20 nm and a molecular mass of 756 kDa and binds more than 60 chlorophylls. The SBA particles, with average internal pore diameters of 15 nm (SBA(15)) and 23 nm (SBA(23)), adsorbed 4.7 and 15 mg of PSII/g SBA, respectively. Measurement with a confocal laser-scanning microscope indicated the adsorption of PSII to the surface and the inner space of the SBA(23) particles, indicating the adsorption of PSII into the 23 nm silica nanopores. PSII did not bind to the inner pores of SBA(15). PSII bound to SBA(23) showed the high and stable activity of a photosynthetic oxygen-evolving reaction, indicating the light-driven electron transport from water to the quinone molecules added in the outer medium. The PSII-SBA conjugate can be a new material for photosensors and artificial photosynthetic systems.     

Ultrafast time-resolved carotenoid to-bacteriochlorophyll energy transfer in LH2 complexes from photosynthetic bacteria

Steady-state and ultrafast time-resolved optical spectroscopic investigations have been carried out at 293 and 10 K on LH2 pigment-protein complexes isolated from three different strains of photosynthetic bacteria: Rhodobacter (Rb.) sphaeroides G1C, Rb. sphaeroides 2.4.1 (anaerobically and aerobically grown), and Rps. acidophila 10050. The LH2 complexes obtained from these strains contain the carotenoids, neurosporene, spheroidene, spheroidenone, and rhodopin glucoside, respectively. These molecules have a systematically increasing number of pi-electron conjugated carbon-carbon double bonds. Steady-state absorption and fluorescence excitation experiments have revealed that the total efficiency of energy transfer from the carotenoids to bacteriochlorophyll is independent of temperature and nearly constant at approximately 90% for the LH2 complexes containing neurosporene, spheroidene, spheroidenone, but drops to approximately 53% for the complex containing rhodopin glucoside. Ultrafast transient absorption spectra in the near-infrared (NIR) region of the purified carotenoids in solution have revealed the energies of the S1 (2(1)Ag-)-->S2 (1(1)Bu+) excited-state transitions which, when subtracted from the energies of the S0 (1(1)Ag-)-->S2 (1(1)Bu+) transitions determined by steady-state absorption measurements, give precise values for the positions of the S1 (2(1)Ag-) states of the carotenoids. Global fitting of the ultrafast spectral and temporal data sets have revealed the dynamics of the pathways of de-excitation of the carotenoid excited states. The pathways include energy transfer to bacteriochlorophyll, population of the so-called S* state of the carotenoids, and formation of carotenoid radical cations (Car*+). The investigation has found that excitation energy transfer to bacteriochlorophyll is partitioned through the S1 (1(1)Ag-), S2 (1(1)Bu+), and S* states of the different carotenoids to varying degrees. This is understood through a consideration of the energies of the states and the spectral profiles of the molecules. A significant finding is that, due to the low S1 (2(1)Ag-) energy of rhodopin glucoside, energy transfer from this state to the bacteriochlorophylls is significantly less probable compared to the other complexes. This work resolves a long-standing question regarding the cause of the precipitous drop in energy transfer efficiency when the extent of pi-electron conjugation of the carotenoid is extended from ten to eleven conjugated carbon-carbon double bonds in LH2 complexes from purple photosynthetic bacteria.     

Unique incorporation behavior of amino acid-type surfactant into phospholipid vesicle membrane

The incorporation behavior of some anionic surfactants, including amino acid-type surfactants, on phospholipid vesicles was investigated. This was done by measuring the release of a vesicle-entrapped fluorescence probe and the scattered light intensities of vesicle particles in the surfactant solution as a function of surfactant concentration and time. Sodium dodecyl sulfate, sodium dodecanesulfonate, sodium dodecanoyl sarcosinate, and sodium dodecanoyl glutamate were employed in this study. All surfactants ruptured the phospholipid vesicle at around each critical micelle concentration by mixed micelle formation with phospholipid. While leakage of the fluorescence probe took place at a very low concentration in the sulfate- or sulfonate-type surfactant systems, it occurred at the concentration just below the CMC in the amino acid-type surfactant systems. Kinetic analysis of the release of the probe from the vesicles showed that the former surfactants adsorbed independently and homogeneously onto the phospholipid vesicles, while the latter surfactants were cooperatively incorporated.     

Site-specific incorporation of the mucin-type N-acetylgalactosamine-alpha-O-threonine into protein in Escherichia coli

Glycosylation is a prevalent posttranslational process capable of augmenting and modulating protein function. Efficient synthesis of high-purity, homogeneous glycoproteins is essential for the study of unique protein glycoforms and for the manufacture of therapeutically relevant forms. A promising new strategy for controlled in vivo synthesis of glycoproteins was recently established using suppressor tRNA technology. Using an evolved tRNA aminoacyl synthetase-tRNA pair from Methanococcus jannaschii, the glycosyl amino acid, N-acetylglucosamine-beta-O-serine (GlcNAc-beta-Ser), was site-specifically introduced into proteins cotranslationally in Escherichia coli. Herein, we report the evolution of a new tRNA aminoacyl synthetase-tRNA pair that has expanded the repertoire of glycoproteins that can be expressed in E. coli to contain the other major O-linked glycan, N-acetylgalactosamine-alpha-O-threonine (GalNAc-a-Thr).     

Solidification and melting of cetane confined in the nanopores of silica gel

Synthetic silica gels with six different effective diameters of nano-pores (3–30 nm) were loaded with n-hexadecane (cetane) after the elimination of adsorbed water. Kinetics of the solidification and melting of cetane was studied by differential scanning calorimetry (DSC) above the room temperature. Two thermodynamically different states of cetane were found in the samples: the free (bulk)-cetane state and the confined-cetane state. As suspected, the third state of cetane can be amorphous. This has been indicated by the small total transformation heat. The complex crystallization effect of cetane has been found to obey the nucleation-and-growth kinetics and also to depend on the dimensions of confining pores of silica gel. The melting of cetane seems to vary only with the average diameter of silica gel pores, which satisfies the Gibbs–Thompson relation. The presented results validate the applicability of the DSC technique for the porometry. The cetane-medium calibration curve for the silica gel nano-thermoporometry has been determined.