Rob B. M. Koehorst

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. With the use of target analysis, we managed to spectrally resolve seven different pigment pools in the phycobilisomes and photosystems I and II, and to determine the rates of excitation energy transfer between them. In addition, the fraction of quenched phycobilisomes and the rates of charge separation and quenching were resolved. Under our illumination conditions, ∼72% of the phycobilisomes in OverOCP appeared to be substantially quenched. For wild-type cells, this number was only ∼29%. It is revealed that upon OCP activation, a bilin chromophore in the core of the phycobilisome, here called APC(Q)(660), with fluorescence maximum at 660 nm becomes an effective quencher that prevents more than 80% of the excitations in the phycobilisome to reach Photosystems I and II. The quenching rate of its excited state is extremely fast, that is, at least (∼240 ± 60 fs)(-1). It is concluded that the quenching is most likely caused by charge transfer between APC(Q)(660) and the OCP carotenoid hECN in its activated form.

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. Here we have used spectrally resolved picosecond fluorescence to study wild-type and two mutated PBs. The results demonstrate that the quenching for all types of PBs takes place on an allophycocyanin bilin emitting at 660 nm (APC(Q)(660)) with a molecular quenching rate that is faster than (1 ps)(-1). Moreover, it is concluded that both the mechanism and the site of quenching are the same in vitro and in vivo. Thus, utilization of the in vitro system should make it possible in the future to elucidate whether the quenching is caused by charge transfer between APC(Q)(660) and OCP or by excitation energy transfer from APC(Q)(660) to the S(1) state of the carotenoid--a distinction that is very hard, if not impossible, to make in vivo.

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. The results of the spectroscopic studies conducted in our group compellingly demonstrate a critical role of membrane embedment of the protein both during infectious entry of the virus into the host cell and during assembly of the new virion in the host membrane. The protein is effectively embedded in the membrane by a strong C-terminal interfacial anchor, which together with a simple tilt mechanism and a subtle structural adjustment of the extreme end of its N terminus provides favourable thermodynamical association of the protein in the lipid bilayer. This basic physicochemical rule cannot be violated and any new bionanotechnology that will emerge from bacteriophage M13 should take this into account.

Dependence of M13 Major Coat Protein Oligomerization and Lateral Segregation on Bilayer Composition

M13 major coat protein was derivatized with BODIPY (n-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-yl)methyl iodoacetamide), and its aggregation was studied in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and DOPC/1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (DOPG) or 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)/DOPG (model systems of membranes with hydrophobic thickness matching that of the protein) using photophysical methodologies (time-resolved and steady-state self-quenching, absorption, and emission spectra). It was concluded that the protein is essentially monomeric, even in the absence of anionic phospholipids. The protein was also incorporated in pure bilayers of lipids with a strong mismatch with the protein transmembrane length, 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEuPC, longer lipid) and 1,2-dimyristoleoyl-sn-glycero-3-phosphocholine (DMoPC, shorter lipid), and in lipidic mixtures containing DOPC and one of these lipids. The protein was aggregated in the pure vesicles of mismatching lipid but remained essentially monomeric in the mixtures as detected from BODIPY fluorescence emission self-quenching. From fluorescence resonance energy transfer (FRET) measurements (donor-n-(iodoacetyl)aminoethyl-1-sulfonaphthylamine (IAEDANS)-labeled protein; acceptor-BODIPY labeled protein), it was concluded that in the DEuPC/DOPC and DMoPC/DOPC lipid mixtures, domains enriched in the protein and the matching lipid (DOPC) are formed.

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. When the peptides were inserted into bilayers with different acyl chain lengths or containing different concentrations of cholesterol, small changes in tilt angle were observed as response to hydrophobic mismatch, whereas the rotation angle appeared to be independent of lipid composition. In all cases, the tilt angles were significantly larger than those previously determined from (2)H NMR experiments, supporting recent suggestions that the relatively long timescale of (2)H NMR measurements may result in an underestimation of tilt angles due to partial motional averaging. It is concluded that although the fluorescence technique has a rather low resolution and limited accuracy, it can be used to resolve the discrepancies observed between previous (2)H NMR experiments and molecular-dynamics simulations.

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. The steady-state fluorescence spectra were analyzed using a three-component spectral model that enabled the separation of Stokes shift contributions from water and internal label dynamics, and protein topology. We found that most of the fluorescence originated from BADAN labels that were hydrogen-bonded to water molecules even within the hydrophobic core of the membrane. Our spectral decomposition method revealed the embedment and topology of the labeled protein in the membrane bilayer under various conditions of headgroup charge and lipid chain length, as well as key characteristics of the membrane such as hydration level and local polarity, provided by the local dielectric constant.

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. This component decays much more rapidly (t(1/2) approximately 2 ps) in the case of the Ca(2+)-discharged obelin than aequorin (t(1/2) approximately 30 ps). The second component at lower energy shows several intermediates in the 150-500 ps times, with a final species having spectral maxima 19 400 cm(-1), bound to Ca(2+)-discharged obelin, and 21 300 cm(-1), bound to Ca(2+)-discharged aequorin, and both have a fluorescence decay lifetime of 4 ns. It is proposed that the rapid kinetics of these fluorescence transients on the picosecond time scale, correspond to times for relaxation of the protein structural environment of the binding cavity.

Fluorescence detected magnetic resonance (FDMR) spectroscopy of chlorophyll-proteins from barley

Fluorescence detected magnetic resonance (FDMR) spectra and fluorescence emission spectra at 4.2 K of chlorophyll-proteins isolated and purified from barley thylakoids are presented. The FDMR spectra show the occurrence of chlorophylla triplet states in all five chlorophyll-proteins studied, namely Chla-P1, Chla-P2, Chla-P3, Chla/b-P1 and Chla/b-P2.The presence of more than one chlorophyll triplet each associated with a chlorophyll emitting at a specific wavelength gives rise to a characteristic wavelength dependence of the FDMR spectrum of chlorophyll-proteins. The zero field splitting parameters measured, combined with the observed fluorescence emission wavelengths suggest that three types of interactions of the Mg atom of chlorophylla occur in these proteins: a type similar to that in the parallel dimer (Chla·H2O)2, seen at 721 nm for Chla-P1 leading to a positive FDMR signal; a type like that in Chla· 2 pyridine also giving a positive FDMR signal, seen in Chla-P2 and Chla-P3; and a third type similar to that in Chla· 2H2O leading to a negative FDMR signal, seen for Chla-P1 at 679 nm, and for Chla/b-P1 and Chla/b-P2.The FDMR spectrum in the antenna of photosystem I (Chla-P1) can probably be ascribed to that of a trap formed by a pair of interacting chlorophylla molecules, indicating that the organisation of chlorophyll in the antenna may not in principle be very different from that in the photosystem I reaction centre, and that it contains approximately plane-parallel chlorophylla pairs. Chla-P2 and Chla-P3 do not show a long wavelength (>700 nm) emission, suggesting a much weaker interaction between chlorophyll molecules in these proteins compared to that in Chla-P1. For Chla/b-P1 and Chla/b-P2 the absence of a long wavelength emission and the observation of zero field splitting (ZFS) parameters similar to that of monomeric Chla·2H2O both indicate the absence of strong interactions between chlorophylla molecules in these proteins also, and it is suggested that chlorophylla and chlorophyllb molecules occur in interacting pairs.

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. It is demonstrated that the degree and way of incorporation differs for most lipid phases--upon selective excitation at 570 nm, the amplitude of the fluorescence component that corresponds to membrane-incorporated MC540 is about 20% in gel-, 60% in rippled gel-, and 90% in liquid-crystalline and inverted hexagonal phase, respectively. For thylakoids, the data reveal hindered incorporation of MC540 (amplitude about 30% at 7 degrees C) and marked spectral heterogeneity at all temperatures. The incorporation of MC540 in thylakoids strongly depends on temperature. Remarkably, above 25 degrees C MC540 becomes almost completely extruded from the lipid environment, indicating major rearrangements in the membrane.

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. Based on FRET-derived constraints, a straight α-helix is proposed as the membrane-bound conformation of the coat protein. Different models were tested to represent the molecular conformations of the donor and acceptor moieties. The best model was used to make a quantitative comparison of the FRET data to the structures of M13 coat protein and related coat proteins in the Protein Data Bank. This shows that the membrane-bound conformation of the coat protein is similar to the structure of the coat protein in the bacteriophage that was obtained from x-ray diffraction. Coat protein embedded in stacked, oriented bilayers and in micelles turns out to be strongly affected by the environmental stress of these membrane-mimicking environments. Our findings emphasize the need to study membrane proteins in a suitable environment, such as in fully hydrated unilamellar vesicles. Although larger proteins than M13 major coat protein may be able to handle environmental stress in a different way, any membrane protein with water exposed parts in the C or N termini and hydrophilic loop regions should be treated with care.