Jens Dittmer

The tetra-manganese complex of photosystem II during its redox cycle - X-ray absorption results and mechanistic implications.

Using X-ray absorption spectroscopy (XAS), relevant information on structure and oxidation state of the water-oxidizing Mn complex of photosystem II has been obtained for all four semi-stable intermediate states of its catalytic cycle. We summarize our recent XAS results and discuss their mechanistic implications. The following aspects are covered: (a) information content of X-ray spectra (pre-edge feature, edge position, extended X-ray absorption fine-structure (EXAFS), dichroism in the EXAFS of partially oriented samples); (b) S(1)-state structure; (c) X-ray edge results on oxidation state changes; (d) EXAFS results on structural changes during the S-state cycle; (e) a structural model for the Mn complex in its S(3)-state; (f) XAS-based working model for the S(2)-S(3) transition; (g) XAS-based working model for the S(0)-S(1) transition; (h) potential role of hydrogen atom abstraction by the Mn complex. Finally, we present a specific hypothesis on the mechanism of dioxygen formation during the S(3)-(S(4))-S(0) transition. According to this hypothesis, water oxidation is facilitated by manganese reduction that is coupled to proton transfer from a substrate water to bridging oxides.

Incorporation of antimicrobial peptides into membranes: a combined liquid-state NMR and molecular dynamics study of alamethicin in DMPC/DHPC bicelles.

Detailed insight into the interplay between antimicrobial peptides and biological membranes is fundamental to our understanding of the mechanism of bacterial ion channels and the action of these in biological host-defense systems. To explore this interplay, we have studied the incorporation, membrane-bound structure, and conformation of the antimicrobial peptide alamethicin in lipid bilayers using a combination of 1H liquid-state NMR spectroscopy and molecular dynamics (MD) simulations. On the basis of experimental NMR data, we evaluate simple in-plane and transmembrane incorporation models as well as pore formation for alamethicin in DMPC/DHPC (1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine/1,2-dihexanoyl-sn-glycero-3-phosphatidylcholine) bicelles. Peptide-lipid nuclear Overhauser effect (NOE) and paramagnetic relaxation enhancement (PRE) data support a transmembrane configuration of the peptide in the bilayers, but they also reveal that the system cannot be described by a single simple conformational model because there is a very high degree of dynamics and heterogeneity in the three-component system. To explore the origin of this heterogeneity and dynamics, we have compared the NOE and PRE data with MD simulations of an ensemble of alamethicin peptides in a DMPC bilayer. From all-atom MD simulations, the contacts between peptide, lipid, and water protons are quantified over a time interval up to 95 ns. The MD simulations provide a statistical base that reflects our NMR data and even can explain some initially surprising NMR results concerning specific interactions between alamethicin and the lipids.

Stepwise Transition of the Tetra-Manganese Complex of Photosystem II to a Binuclear Mn2(μ-O)2 Complex in Response to a Temperature Jump: A Time-Resolved Structural Investigation Employing X-Ray Absorption Spectroscopy

In oxygenic photosynthesis, water is oxidized at a protein-cofactor complex comprising four Mn atoms and, presumably, one calcium. Using multilayers of Photosystem II membrane particles, we investigated the time course of the disassembly of the Mn complex initiated by a temperature jump from 25 degrees C to 47 degrees C and terminated by rapid cooling after distinct heating periods. We monitored polarographically the oxygen-evolution activity, the amount of the Y(D)(ox) radical and of released Mn(2+) by EPR spectroscopy, and the structure of the Mn complex by x-ray absorption spectroscopy (XAS, EXAFS). Using a novel approach to analyze time-resolved EXAFS data, we identify three distinct phases of the disassembly process: (1) Loss of the oxygen-evolution activity and reduction of Y(D)(ox) occur simultaneously (k(1) = 1.0 min(-1)). EXAFS spectra reveal the concomitant loss of an absorber-backscatterer interaction between heavy atoms separated by approximately 3.3 A, possibly related to Ca release. (2) Subsequently, two Mn(III) or Mn(IV) ions seemingly separated by approximately 2.7 A in the native complex are reduced to Mn(II) and released (k(2) = 0.18 min(-1)). The x-ray absorption spectroscopy data is highly suggestive that the two unreleased Mn ions form a di- micro -oxo bridged Mn(III)(2) complex. (3) Finally, the tightly-bound Mn(2)( micro -O)(2) unit is slowly reduced and released (k(3) = 0.014 min(-1)).

Bromine K-edge EXAFS studies of bromide binding to bromoperoxidase from Ascophyllum nodosum

Bromine K‐edge EXAFS studies have been carried out for bromide/peroxidase samples in Tris buffer at pH 8. The results are compared with those of aqueous (Tris‐buffered) bromide and vanadium model compounds containing Br‐V, Br‐C(aliphatic) and Br‐C(aromatic) bonds. It is found that bromide does not coordinate to the vanadium centre. Rather, bromine binds covalently to carbon. A possible candidate is active site serine.

An Unusual Intrinsically Disordered Protein from the Model Legume Lotus japonicus Stabilizes Proteins in Vitro

Intrinsic structural disorder is a prevalent feature of proteins with chaperone activity. Using a complementary set of techniques, we have structurally characterized LjIDP1 (intrinsically disordered protein 1) from the model legume Lotus japonicus, and our results provide the first structural characterization of a member of the Lea5 protein family (PF03242). Contrary to in silico predictions, we show that LjIDP1 is intrinsically disordered and probably exists as an ensemble of conformations with limited residual β-sheet, turn/loop, and polyproline II secondary structure. Furthermore, we show that LjIDP1 has an inherent propensity to undergo a large conformational shift, adopting a largely α-helical structure when it is dehydrated and in the presence of different detergents and alcohols. This is consistent with an overrepresentation of order-promoting residues in LjIDP1 compared with the average of intrinsically disordered proteins. In line with functioning as a chaperone, we show that LjIDP1 effectively prevents inactivation of two model enzymes under conditions that promote protein misfolding and aggregation. The LjIdp1 gene is expressed in all L. japonicus tissues tested. A higher expression level was found in the root tip proximal zone, in roots inoculated with compatible endosymbiotic M. loti, and in functional nitrogen-fixing root nodules. We suggest that the ability of LjIDP1 to prevent protein misfolding and aggregation may play a significant role in tissues, such as symbiotic root nodules, which are characterized by high metabolic activity.

Early Stages of Amyloid Fibril Formation Studied by Liquid-State NMR: The Peptide Hormone Glucagon

The 29-residue peptide hormone glucagon forms amyloid fibrils within a few hours at low pH. In this study, we use glucagon as a model system to investigate fibril formation by liquid-state (1)H-NMR spectroscopy One-dimensional, correlation, and diffusion experiments monitoring the fibril formation process provide insight into the early stages of the pathway on which the molecules aggregate to fibrils. In conjunction with these techniques, exchange experiments give information about the end-state conformation. Within the limits of detection, there are no signs of larger oligomeric intermediates in the course of the fibril formation process. Kinetic information is extracted from the time course of the residual free glucagon signal decay. This suggests that glucagon amyloids form by a nucleated growth mechanism in which trimers (rather than monomers) of glucagon interact directly with the growing fibrils rather than with each other. The results of proton/deuterium exchange experiments on mature fibrils with subsequent dissolution show that the N-terminal of glucagon is the least amenable to exchange, which indicates that this part is strongly involved in the intermolecular bonds of the fibrils.

NMR Solution Structure, Backbone Mobility, and Homology Modeling of c-Type Cytochromes from Gram-Positive Bacteria

The solution structure of oxidized cytochrome c553 (71 amino acid residues) from the Gram‐positive bacterium Bacillus pasteurii is here reported and compared with the available crystal structure. The solution structure is obtained from 1609 meaningful NOE data (22.7 per residue), 76 dihedral angles, and 59 pseudocontact shifts. The root mean square deviations from the average structure are 0.25±0.07 and 0.59±0.13 Å for the backbone and all heavy atoms, respectively, and the quality assessment of the structure is satisfactory. The solution structure closely reproduces the fold observed in the crystal structure. The backbone mobility was then investigated through amide 15N relaxation rate and 15N–1H NOE measurements. The protein is rigid in both the sub‐nanosecond and millisecond time scales, probably due to the relatively large heme:number of amino acids ratio. Modeling of eight c‐type cytochromes from other Gram‐positive bacteria with a high sequence identity (>30 %) to the present cytochrome c553 was performed. Analysis of consensus features accounts for the relatively low reduction potential as being due to extensive heme hydration and indicates residues 34–35, 44–46, 69–72, and 75 as a conserved hydrophobic patch for the interaction with a protein partner. At variance with mitochondrial c‐type cytochrome, this protein does not experience pH‐dependent coordination equilibria. The reasons for this difference are analyzed.

Marennine, Promising Blue Pigments from a Widespread Haslea Diatom Species Complex

In diatoms, the main photosynthetic pigments are chlorophylls a and c, fucoxanthin, diadinoxanthin and diatoxanthin. The marine pennate diatom Haslea ostrearia has long been known for producing, in addition to these generic pigments, a water-soluble blue pigment, marennine. This pigment, responsible for the greening of oysters in western France, presents different biological activities: allelopathic, antioxidant, antibacterial, antiviral, and growth-inhibiting. A method to extract and purify marennine has been developed, but its chemical structure could hitherto not be resolved. For decades, H. ostrearia was the only organism known to produce marennine, and can be found worldwide. Our knowledge about H. ostrearia-like diatom biodiversity has recently been extended with the discovery of several new species of blue diatoms, the recently described H. karadagensis, H. silbo sp. inedit. and H. provincialis sp. inedit. These blue diatoms produce different marennine-like pigments, which belong to the same chemical family and present similar biological activities. Aside from being a potential source of natural blue pigments, H. ostrearia-like diatoms thus present a commercial potential for aquaculture, cosmetics, food and health industries.

Solid-State NMR Correlation Experiments and Distance Measurements in Paramagnetic Metalorganics Exemplified by Cu-Cyclam

We show how to record and analyze solid-state NMR spectra of organic paramagnetic complexes with moderate hyperfine interactions using the Cu-cyclam complex as an example. Assignment of the (13)C signals was performed with the help of density functional theory (DFT) calculations. An initial assignment of the (1)H signals was done by means of (1)H-(13)C correlation spectra. The possibility of recording a dipolar HSQC spectrum with the advantage of direct (1)H acquisition is discussed. Owing to the paramagnetic shifting the resolution of such paramagnetic (1)H spectra is generally better than for diamagnetic solid samples, and we exploit this advantage by recording (1)H-(1)H correlation spectra with a simple and short pulse sequence. This experiment, along with a Karplus relation, allowed for the completion of the (1)H signal assignment. On the basis of these data, we measured the distances of the carbon atoms to the copper center in Cu-cyclam by means of (13)CR2 relaxation experiments combined with the electronic relaxation determined by EPR.

Similarities between intra- and intermolecular hydrogen bonds in RNA kissing complexes found by means of cross-correlated relaxation

The bond lengths and dynamics of intra- and intermolecular hydrogen bonds in an RNA kissing complex have been characterized by determining the NMR relaxation rates of various double- and triple-quantum coherences that involve an imino proton and two neighboring nitrogen-15 nuclei belonging to opposite bases. New experiments allow one to determine the chemical shift anisotropy of the imino protons. The bond lengths derived from dipolar relaxation and the lack of modulations of the nitrogen chemical shifts indicate that the intermolecular hydrogen bonds which hold the kissing complex together are very similar to the intramolecular hydrogen bonds in the double-stranded stem of the RNA.