Efficiency of excitation energy trapping in the green photosynthetic bacterium Chlorobaculum tepidum.

During the millions of years of evolution, photosynthetic organisms have adapted to almost all terrestrial and aquatic habitats, although some environments are obviously more suitable for photosynthesis than others. Photosynthetic organisms living in low-light conditions require on the one hand a large light-harvesting apparatus to absorb as many photons as possible. On the other hand, the excitation trapping time scales with the size of the light-harvesting system, and the longer the distance over which the formed excitations have to be transferred, the larger the probability to lose excitations.

Light harvesting and blue-green light induced non-photochemical quenching in two different C-phycocyanin mutants of Synechocystis PCC 6803.

Cyanobacteria are oxygen-evolving photosynthetic organisms that harvest sunlight and convert excitation energy into chemical energy. Most of the light is absorbed by large light harvesting complexes called phycobilisomes (PBs). In high-light conditions, cyanobacteria switch on a photoprotective mechanism called non-photochemical quenching (NPQ): During this process, absorption of blue-green light transforms the inactive orange form of the orange carotenoid protein OCP (OCP(o)) into the red active form OCP(r) that subsequently binds to the PB, resulting in a substantial loss of excitation energy and corresponding decrease of the fluorescence.

Picosecond kinetics of light harvesting and photoprotective quenching in wild-type and mutant phycobilisomes isolated from the cyanobacterium Synechocystis PCC 6803.

In high light conditions, cyanobacteria dissipate excess absorbed energy as heat in the light-harvesting phycobilisomes (PBs) to protect the photosynthetic system against photodamage. This process requires the binding of the red active form of the Orange Carotenoid Protein (OCP(r)), which can effectively quench the excited state of one of the allophycocyanin bilins. Recently, an in vitro reconstitution system was developed using isolated OCP and isolated PBs from Synechocystis PCC 6803.

Time-resolved fluorescence and fluorescence anisotropy of fluorescein-labeled poly(N-isopropylacrylamide) incorporated in polymersomes.

The phase behavior of fluorescein isothiocyanate (FITC) labeled poly(N-isopropylacrylamide) (PNIPAAm) incorporated in polymersomes (Ps) was studied by monitoring the fluorescence lifetime (FL) and the time-resolved fluorescence anisotropy (TRFA) as a function of temperature at pH 7.4. Ps containing FITC-labeled PNIPAAm with a diameter less than 200 nm were prepared by injecting a THF solution of poly(ethylene glycol)-b-poly(d,l-lactide) (mPEG-PDLLA) and FITC tagged PNIPAAm (FITC-N) into phosphate buffered saline (PBS, pH 7.

Site, rate, and mechanism of photoprotective quenching in cyanobacteria.

In cyanobacteria, activation of the Orange Carotenoid Protein (OCP) by intense blue-green light triggers photoprotective thermal dissipation of excess absorbed energy leading to a decrease (quenching) of fluorescence of the light harvesting phycobilisomes and, concomitantly, of the energy arriving to the reaction centers. Using spectrally resolved picosecond fluorescence, we have studied cells of wild-type Synechocystis sp. PCC 6803 and of mutants without and with extra OCP (ΔOCP and OverOCP) both in the unquenched and quenched state.

Tilt and rotation angles of a transmembrane model peptide as studied by fluorescence spectroscopy.

In this study the membrane orientation of a tryptophan-flanked model peptide, WALP23, was determined by using peptides that were labeled at different positions along the sequence with the environmentally sensitive fluorescent label BADAN. The fluorescence properties, reflecting the local polarity, were used to determine the tilt and rotation angles of the peptide based on an ideal alpha-helix model. For WALP23 inserted in dioleoylphosphatidylcholine (DOPC), an estimated tilt angle of the helix with respect to the bilayer normal of 24 degrees +/- 5 degrees was obtained.

Picosecond fluorescence relaxation spectroscopy of the calcium-discharged photoproteins aequorin and obelin.

Addition of calcium ions to the Ca(2+)-regulated photoproteins, such as aequorin and obelin, produces a blue bioluminescence originating from a fluorescence transition of the protein-bound product, coelenteramide. The kinetics of several transient fluorescent species of the bound coelenteramide is resolved after picosecond-laser excitation and streak camera detection. The initially formed spectral distributions at picosecond-times are broad, evidently comprised of two contributions, one at higher energy (approximately 25,000 cm(-1)) assigned as from the Ca(2+)-discharged photoprotein-bound coelenteramide in its neutral state.

Profiling of dynamics in protein-lipid-water systems: a time-resolved fluorescence study of a model membrane protein with the label BADAN at specific membrane depths.

Profiles of lipid-water bilayer dynamics were determined from picosecond time-resolved fluorescence spectra of membrane-embedded BADAN-labeled M13 coat protein. For this purpose, the protein was labeled at seven key positions. This places the label at well-defined locations from the water phase to the center of the hydrophobic acyl chain region of a phospholipid model membrane, providing us with a nanoscale ruler to map membranes.

Asymmetric dipping of bacteriophage M13 coat protein with increasing lipid bilayer thickness.

Knowledge about the vertical movement of a protein with respect to the lipid bilayer plane is important to understand protein functionality in the biological membrane. In this work, the vertical displacement of bacteriophage M13 major coat protein in a lipid bilayer is used as a model system to study the molecular details of its anchoring mechanism in a homologue series of lipids with the same polar head group but different hydrophobic chain length. The major coat proteins were reconstituted into 14:1PC, 16:1PC, 18:1PC, 20:1PC, and 22:1PC bilayers, and the fluorescence spectra were measured of the intrinsic tryptophan at position 26 and BADAN attached to an introduced cysteine at position 46, located at the opposite ends of the transmembrane helix.

Viruses: incredible nanomachines. New advances with filamentous phages.

During recent decades, bacteriophages have been at the cutting edge of new developments in molecular biology, biophysics, and, more recently, bionanotechnology. In particular filamentous viruses, for example bacteriophage M13, have a virion architecture that enables precision building of ordered and defect-free two and three-dimensional structures on a nanometre scale. This could not have been possible without detailed knowledge of coat protein structure and dynamics during the virus reproduction cycle.

Conformational studies of peptides representing a segment of TM7 from H+-VO-ATPase in SDS micelles.

The conformation of a transmembrane peptide, sMTM7, encompassing the cytoplasmic hemi-channel domain of the seventh transmembrane section of subunit a from V-ATPase from Saccharomyces cerevisiae solubilized in SDS solutions was studied by circular dichroism (CD) spectroscopy and fluorescence spectroscopy of the single tryptophan residue of this peptide. The results show that the peptide adopts an alpha-helical conformation or aggregated beta-sheet depending on the peptide-to-SDS ratio used. The results are compared with published data about a longer version of the peptide (i.

Exploring the structure of the N-terminal domain of CP29 with ultrafast fluorescence spectroscopy.

A high-throughput Förster resonance energy transfer (FRET) study was performed on the approximately 100 amino acids long N-terminal domain of the photosynthetic complex CP29 of higher plants. For this purpose, CP29 was singly mutated along its N-terminal domain, replacing one-by-one native amino acids by a cysteine, which was labeled with a BODIPY fluorescent probe, and reconstituted with the natural pigments of CP9, chlorophylls and xanthophylls. Picosecond fluorescence experiments revealed rapid energy transfer (approximately 20-70 ps) from BODIPY at amino-acid positions 4, 22, 33, 40, 56, 65, 74, 90, and 97 to Chl a molecules in the hydrophobic part of the protein.

From 'I' to 'L' and back again: the odyssey of membrane-bound M13 protein.

The major coat protein of the filamentous bacteriophage M13 is a surprising protein because it exists both as a membrane protein and as part of the M13 phage coat during its life cycle. Early studies showed that the phage-bound structure of the coat protein was a continuous I-shaped alpha-helix. However, throughout the years various structural models, both I-shaped and L-shaped, have been proposed for the membrane-bound state of the coat protein.

Temperature dependence of the lipid packing in thylakoid membranes studied by time- and spectrally resolved fluorescence of Merocyanine 540.

The lipid packing of thylakoid membranes is an important factor for photosynthetic performance. However, surprisingly little is known about it and it is generally accepted that the bulk thylakoid lipids adopt the liquid-crystalline phase above -30 degrees C and that a phase transition occurs only above 45 degrees C. In order to obtain information on the nature of the lipid microenvironment and its temperature dependence, steady-state and time-resolved fluorescence measurements were performed on the fluorescence probe Merocyanine 540 (MC540) incorporated in isolated spinach thylakoids and in model lipid systems (dipalmitoyl phosphatidylcholine and dioleoyl phosphatidylethanolamine) adopting different phases.

Site-directed fluorescence labeling of a membrane protein with BADAN: probing protein topology and local environment.

The work presented here describes a new and simple method based on site-directed fluorescence labeling using the BADAN label that permits the examination of protein-lipid interactions in great detail. We applied this technique to a membrane-embedded, mainly alpha-helical reference protein, the M13 major coat protein. Using a high-throughput approach, 40 site-specific cysteine mutants were prepared of the 50-residues long protein.

Solubilization of V-ATPase transmembrane peptides by amphipol A8-35.

Two transmembrane peptides encompassing the seventh transmembrane section of subunit a from V-ATPase from Saccharomyces cerevisiae were studied as complexes with APols A8-35 by CD and fluorescence spectroscopy, with the goal to use APols to provide a membrane-mimicking environment for the peptides. CD spectroscopy was used to obtain the overall secondary structure of the peptides, whereas fluorescence spectroscopy provided information about the local environment of their tryptophan residues. The fluorescence results indicate that both peptides are trapped by APols and the CD results that they adopt a beta-sheet conformation.

Structure of membrane-embedded M13 major coat protein is insensitive to hydrophobic stress.

The structure of a membrane-embedded alpha-helical reference protein, the M13 major coat protein, is characterized under different conditions of hydrophobic mismatch using fluorescence resonance energy transfer in combination with high-throughput mutagenesis. We show that the structure is similar in both thin (14:1) and thick (20:1) phospholipid bilayers, indicating that the protein does not undergo large structural rearrangements in response to conditions of hydrophobic mismatch. We introduce a "helical fingerprint" analysis, showing that amino acid residues 1-9 are unstructured in both phospholipid bilayers.

FRET study of membrane proteins: determination of the tilt and orientation of the N-terminal domain of M13 major coat protein.

A formalism for membrane protein structure determination was developed. This method is based on steady-state FRET data and information about the position of the fluorescence maxima on site-directed fluorescent labeled proteins in combination with global data analysis utilizing simulation-based fitting. The methodology was applied to determine the structural properties of the N-terminal domain of the major coat protein from bacteriophage M13 reconstituted into unilamellar DOPC/DOPG (4:1 mol/mol) vesicles.

Spectroscopy and photophysics of self-organized zinc porphyrin nanolayers. 2. Transport properties of singlet excitation.

Exciton diffusion has been studied in 5-25-nm-thick films of zinc tetra-(p-octylphenyl)-porphyrin (ZnTOPP) spin-coated onto quartz slides by intentional doping with quenchers using steady-state as well as time-resolved fluorescence spectroscopy. The fluorescence spectra of the films are very similar to those of solutions, indicating emission from localized exciton states. From the dependence of the fluorescence quenching on the quencher concentration and fluorescence lifetime measurements, the exciton diffusion can be concluded to be quasi-one-dimensional with an exciton diffusion length of 9 +/- 3 nm and an intrastack energy-transfer rate constant of 10(11)-10(12) s(-1).

Spectroscopy and photophysics of self-organized zinc porphyrin nanolayers. 1. Optical spectroscopy of excitonic interactions involving the soret band.

The photophysical properties of excited singlet states of zinc tetra-(p-octylphenyl)-porphyrin in 5-25-nm-thick films spin-coated onto quartz slides have been investigated by optical spectroscopy. Analysis of the polarized absorption spectra using a dipole-dipole exciton model with two mutually perpendicular transition dipole moments per molecule shows that the films are built from linear aggregates, i.e.

FRET study of membrane proteins: simulation-based fitting for analysis of membrane protein embedment and association.

A new formalism for the simultaneous determination of the membrane embedment and aggregation of membrane proteins is developed. This method is based on steady-state Förster (or fluorescence) resonance energy transfer (FRET) experiments on site-directed fluorescence labeled proteins in combination with global data analysis utilizing simulation-based fitting. The simulation of FRET was validated by a comparison with a known analytical solution for energy transfer in idealized membrane systems.

Membrane-bound peptides mimicking transmembrane Vph1p helix 7 of yeast V-ATPase: a spectroscopic and polarity mismatch study.

The V-ATPases are a family of ATP-dependent proton pumps, involved in a variety of cellular processes, including bone breakdown. V-ATPase enzymes that are too active in the latter process can result in osteoporosis, and inhibitors of the enzyme could be used to treat this disease. As a first step in studying the structure and function of the membrane-embedded interface at which proton translocation takes place, and its role in V-ATPase inhibition, synthetic peptides P1 and P2 consisting of 25 amino acid residues are presented here that mimic Vph1p helix 7 of yeast V-ATPase.

Membrane-bound conformation of M13 major coat protein: a structure validation through FRET-derived constraints.

M13 major coat protein, a 50-amino-acid-long protein, was incorporated into DOPC/DOPG (80/20 molar ratio) unilamellar vesicles. Over 60% of all amino acid residues was replaced with cysteine residues, and the single cysteine mutants were labeled with the fluorescent label I-AEDANS. The coat protein has a single tryptophan residue that is used as a donor in fluorescence (or Förster) resonance energy transfer (FRET) experiments, using AEDANS-labeled cysteines as acceptors.

Spectroscopic studies of oligodiacetylenes in solution and polymer film.

The photophysical properties of a series of monomeric, dimeric and trimeric oligodiacetylenes (ODAs; oligoenynes) bearing trimethylsilyl, t-butyl and n-octyl end-capping substituents were studied in solution and in a polymer film. Emission studies show a significant emission of oligodiacetylenes in solution, which increased with increased conjugation. This is remarkable given the near absence of fluorescence in oligoenes of similar length (e.

Lipid bilayer topology of the transmembrane alpha-helix of M13 Major coat protein and bilayer polarity profile by site-directed fluorescence spectroscopy.

This article presents a new formalism to perform a quantitative fluorescence analysis using the Stokes shift of AEDANS-labeled cysteine mutants of M13 major coat protein incorporated in lipid bilayers. This site-directed fluorescence spectroscopy approach enables us to obtain the topology of the bilayer-embedded transmembrane alpha-helix from the orientation and tilt angles, and relative bilayer location. Both in pure dioleoylphosphatidylcholine and dioleoylphosphatidylcholine/dioleoylphosphatidylglycerol (4:1 mol/mol) bilayers, which have a similar bilayer thickness, the tilt angle of the transmembrane helix of the coat protein turns out to be 23 degrees +/- 4.