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The classical pathway of complement is initiated by the C1 complex, a multimolecular protease comprising a recognition subunit (C1q) and two modular serine proteases (C1r and C1s) associated as a Ca2+-dependent tetramer (C1s-C1r-C1r-C1s). Early studies have allowed identification of specialized functional domains in these proteins and have led to low-resolution models of the C1 complex. The objective of current studies is to gain deeper insights into the structure of C1, and the strategy used for this purpose mainly consists of dissecting the C1 components into modular fragments, in order to solve their three-dimensional structure and establish the structural correlates of their function. The aim of this article is to provide an overview of the structural and functional information generated by this approach, with particular emphasis on the domains involved in the assembly, the recognition function, and the highly specific proteolytic properties of C1.

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Substance P, one of the mammalian tachykinins, is known to interact strongly with lipid bilayers and this interaction may play a role in the receptor-peptide recognition process. The conformation of substance P bound to vesicles consisting of perdeuterated phosphatidylcholine has been investigated by means of two-dimensional transferred nuclear Overhauser (trNOE) spectroscopy. Nuclear magnetic resonance data analysis resulted in a unique conformational family characterized by a well-defined conformation of the last seven C-terminal amino acids, which consists of a sequence of nonstandard turns following each other in a helix-like manner. The absence of short- or medium-range trNOE in the N-terminal part indicates its structural flexibility.

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The most effective treatment for pericardial effusion and cardiac tamponade is removal of the pericardial fluid. Surgical pericardiotomy is associated with high mortality and morbidity. Similarly, subcostal percutaneous blind pericardiocentesis was reported to have unacceptably high mortality and complication rates. Major complications associated with blind needle punctures are right heart penetration, hemopericardium, puncture of the coronary arteries, liver and lung bleeding. Even under fluoroscopic guidance and electrocardiographic needle monitoring high complication rates persist. Pericardial drainage has been often inadequate, with frequent recurrences of significant pericardial effusions. Two-dimensional echocardiographically guided pericardiocentesis is reported to improve efficacy and safety of percutaneous puncture. Moreover, it allows immediate verification of the procedural success. We evaluated the efficacy and safety of an echocardiographically guided contrast agent controlled pericardiocentesis. This is a retrospective, descriptive study on 126 consecutive patients who underwent percutaneous pericardiocentesis at the University Hospital Essen, Germany, from 1995 to June 2000. There were 51 women (41%) and 75 men (55%) with a mean age of 52 +/- 14 years. Standard techniques for quantification of pericardial effusion were used. Depending on the localization of the pericardial effusion an apical or subxiphoidal approach was chosen. The puncture was performed under echocardiographic guidance and the position of the needle was controlled by injection of contrast agent. Over a long guidewire a pigtail catheter was inserted through a sheath for further drainage of pericardial fluid. The catheter was removed after a maximum of 48 hours to avoid infection of the pericardial cavity. An apical approach was chosen in 98 patients (78%), a subcostal in 28 patients (22%). The procedure was successful in 99% of the attempts. No death or clinical complication occurred. The maximal pericardial diameter measured by two-dimensional echocardiography was 32 +/- 16 mm before and 5.3 +/- 2 mm after drainage. The calculated pericardial effusion was 657 +/- 342 ml. A fluid volume of 605 +/- 342 ml could be drained. In all patients a pericardial catheter was placed for 1.4 +/- 0.8 days. Recurrence of pericardial effusion occurred in 18 patients (14%). Of these, 15 patients underwent repeated successful pericardiocentesis (2.5 +/- 0.8), and 3 patients were referred to surgical pericardiotomy. Pericardiocentesis under echocardiographic contrast agent guidance is a safe, successful and cost effective procedure for diagnostic and therapeutic drainage of pericardial effusion. Two-dimensional echocardiography allows localization of the optimal puncture site as well as the quantification of the effusion depth. The injection of contrast agents into the pericardial cavity improves the safety and accuracy of the procedure. Even recurrent pericardial effusions can be treated successfully.

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C1r and C1s, the proteases responsible for activation and proteolytic activity of the C1 complex of complement, share similar overall structural organizations featuring five nonenzymic protein modules (two CUB modules surrounding a single EGF module, and a pair of CCP modules) followed by a serine protease domain. Besides highly specific proteolytic activities, both proteases exhibit interaction properties associated with their N-terminal regions. These properties include the ability to bind Ca2+ ions with high affinity, to associate with each other within a Ca2+-dependent C1s-C1r-C1r-C1s tetramer, and to interact with C1q upon C1 assembly. Precise functional mapping of these regions has been achieved recently, allowing identification of the domains responsible for these interactions, and providing a comprehensive picture of their structure and function. The objective of this article is to provide a detailed and up-to-date overview of the information available on these domains, which are keystones of the assembly of C1, and appear to play an essential role at the interface between the recognition function of C1 and its proteolytic activity.

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C1r and C1s, the enzymes responsible for the activation and proteolytic activity of the C1 complex of complement, are modular serine proteases featuring similar overall structural organizations, yet expressing very distinct functional properties within C1. This review will initially summarize available information on the structure and function of the protein modules and serine protease domains of C1r and C1s. It will then focus on the regions of both proteases involved in: (i) assembly of C1s-C1r-C1r-C1s, the Ca(2+)-dependent tetrameric catalytic subunit of C1; (ii) expression of C1 catalytic activities. Particular emphasis will be aid on recent structural and functional studies that provide new insights into the complex mechanisms involved in the assembly, activation, and proteolytic activity of C1.

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Y64 has been replaced in cytochrome c553 from Desulfovibrio vulgaris Hildenborough by phenylalanine, leucine, valine, serine and alanine residues. An NMR study of structural variation induced in both oxidoreduction states of the molecule has been carried out by analysing observed chemical-shift variations. Dynamic changes were evidenced using NH exchange. We have observed that the substitution has a drastic effect on the stability of the molecule in the reduced state, although there is no effect on the reduction potential of the cytochrome. Y64-->F substitution induces particular effects on the NH exchange at the N-terminal, C-terminal and central alpha-helices and increases the stability of the oxidized molecule.

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The calcium-dependent interaction between C1r and C1s, the two homologous serine proteases of the first component of human complement C1, is mediated by their N-terminal regions. The latter comprise an epidermal growth factor (EGF)-like module exhibiting the consensus sequence characteristic of Ca(2+)-binding EGF modules, surrounded by two CUB modules. Due to its Ca2+ binding ability, the C1r EGF-like module (C1r-EGF) is supposed to participate in the C1r-C1s interaction. An additional interesting feature of C1r-EGF is the unusually large loop connecting the first two conserved cysteine residues. The solution structure of synthetic C1r-EGF (residues 123-175) has been determined using nuclear magnetic resonance and combined simulated annealing-restrained molecular dynamics calculations. The resulting family of 19 structures is characterized by a well-ordered C-terminal part (residues Cys 144-Ala174) with a backbone rmsd of 0.7 A and a disordered N-terminal, including the large loop between the first two cysteines (Cys129 and Cys144). This loop is known to be surface exposed and may be expected to participate in domain-domain or protein-protein interactions. In its C-terminal part, C1r-EGF possesses the characteristic EGF fold with a major and a minor beta-sheet. The latter comprises a beta-bulge, and comparison with other EGF-like modules reveals the existence of two distinct structural and sequential motifs in the bulged part. Additional experiments in the presence of 80 mM Ca2+ did not show significant structural variation of C1r-EGF, in keeping with previous observations on blood-clotting factors IX and X.

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C1r is one of the two serine proteases of C1, the first component of complement, in which it is associated in a calcium-dependent manner to the homologous serine protease C1s. This interaction is mediated by the N-terminal region of C1r, which comprises a single epidermal growth factor (EGF)-like module containing the consensus sequence required for calcium binding, surrounded by two CUB modules. With a view to determine the structure of the EGF-like module of C1r and evaluate its contribution to calcium binding, this module [C1r(123-175)] was synthesized by automated solid-phase methodology using the Boc strategy. A first synthesis using the Boc-His(Z) derivative gave very low yield, due to partial deprotection of His residues leading to chain termination by acetylation, and to insertion of glycine residues. This could be circumvented by using the Boc-His(DNP) derivative and by condensation of appropriate glycine-containing segments. The synthetic peptide was efficiently folded under redox conditions to the species with three correct disulfide bridges, as determined by mass spectrometry and N-terminal sequence analyses of thermolytic fragments. The homogeneity of the synthetic peptide was assessed by reversed-phase HPLC and electrospray mass spectrometry. One-dimensional 1H NMR spectroscopic analysis provided evidence that the EGF-like module had a well defined structure, and was able to bind calcium with an apparent Kd of 10 mM. This value, comparable to that found for the isolated EGF-like modules of coagulation factors IX and X, is much higher than that measured for native C1r. As already proposed for factors IX and X, it is suggested that neighbouring module(s), most probably the N-terminal CUB module, contribute(s) to the calcium binding site.

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The solution structure of the Ectothiorhodospira halophila ferrocytochrome c551 has been determined. This molecule belongs to a separate class of small bacterial cytochromes c for which no 3D structure has been reported so far. It is characterized by a very low redox potential (58 mV) and is isolated from the periplasm of halophilic purple phototrophic bacteria. For the 78 residue protein, 1445 NOE derived distance constraints were used in a combined simulated annealing/restrained molecular dynamics calculation. The final ensemble of 37 structures presents a backbone r.m.s.d. of less than 0.5 A compared to the mean structure. The physical viability of these structures was investigated by subjecting eight of them to a constraint free molecular dynamics simulation. No systematic conformational change was observed and the average backbone r.m.s.d. compared to the initial structures was less than 1.5 A. The structure of the E. halophila cytochrome c551 shows a striking resemblance to Azotobacter vinelandii cytochrome c5. Significant differences in backbone conformations occur in three small regions which are implicated in solvent protection of the heme propionates and thiomethyl-8(1). Comparison with Pseudomonas aeruginosa cytochrome c551 reveals that only the common cytochrome c core, i.e. three helices, is conserved. The folding of the protein chain around the heme propionates is very different and results in more efficient solvent protection in Ps. aeruginosa. The electrostatic surface of E. halophila cytochrome c551 was found to be significantly different from mitochondrial cytochromes c and bacterial cytochromes c2 but similar to that of Ps. aeruginosa cytochrome c551.

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In order to study the conformational stability induced by the replacement of Tyr-64 in Desulfovibrio vulgaris Hildenborough (DvH) cytochrome c553, fast peptic digestion of deuterated protein followed by separation and measurement of related peptides using liquid chromatography coupled to electrospray ionization mass spectrometry was performed. We show that the H-bonding and/or solvent accessibility properties were modified by the single-site mutation. The mutant proteins can be classified into two groups: the Y64F and Y64L mutants with nearly unchanged deuterium incorporation compared to the wild-type protein and the Y64S, Y64V and Y64A mutants with increased deuterium incorporation. The 70-74 peptide was the most affected by mutation of Tyr-64, the phenylalanine mutant inducing slight stabilization whereas the serine mutant was significantly destabilized. In addition, from the analysis of the overlapping 37-57 and 38-57 peptides we can conclude that the amide proton of Tyr-38 has been replaced by deuterium in all proteins.

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The lipid composition of Spirulina maxima cells grown in a perdeuterated medium was determined by using nuclear magnetic resonance spectroscopy, fast atom-bombardment-mass spectrometry, gas chromatography-mass spectrometry as well as conventional chemical methods. The extent of deuteration was determined by mass spectrometry and was superior to 97.5%. The major lipids identified in the strain were: non-polar lipids (9%), monogalactosyldiacylglycerol (5%), digalactosyldiacylglycerol (22%), phosphatidylglycerol (31%), sulfoquinovosyldiacylglycerol (32%), phosphatidylinositol (traces). The major fatty acids were 16:0 (80%) and 18:1 (15%). These results demonstrate that the adaptation of the cells to D2O did not imply a profound modification of the lipid composition. The perdeuterated polar lipid mixture dispersed into an excess of water organises spontaneously in a lamellar phase as seen by 31P and deuterium solid state NMR and can therefore be used to prepare perdeuterated model membranes with a well defined composition. Liposomes made using these lipids have a gel to liquid-crystalline phase transition in the range 15-27 degrees C and are in a fluid L alpha phase above this temperature.

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Hydrogen/deuterium exchange as well as charge state distribution monitored by electrospray ionization mass spectrometry were demonstrated to be a powerful and effective new tool for probing conformational properties of proteins in solution. In this paper, the influence of single amino acid replacements on the global conformation of cytochrome C553 from Desulfovibrio vulgaris Hildenborough using isotopic exchange monitored by electrospray ionization mass spectrometry is reported. Based on their respective charge state distributions and isotopic exchanges, we have differentiated relative stability of mutants and a ladder classification with the order being wild-type > Y64F = Y64L > Y64V > Y64A, under specific conditions of pH, is proposed.

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Two-dimensional nuclear magnetic resonance was used to assign the 1H and 13C resonances of ferrocytochrome c-551 from Ectothiorhodospira halophila, a halophilic phototrophic purple bacterium. This 78-residue protein belongs to a small subgroup of class I cytochromes c together with the analogous cytochromes c-551 from E. halochloris and E. abdelmalekii. A nearly complete assignment of 13C resonances was obtained at natural abundance using a gradient-enhanced 1H-detected heteronuclear single quantum coherence experiment (HSQC). This was found to be extremely useful for the unambigous assignment of side chain protons. The secondary structure of the protein was determined from analyses of short- and medium-range nuclear Overhauser enhancements (NOE), amide proton exchange and 13C alpha chemical shifts. Three helices could be identified which are well conserved among the class I cytochromes c. There is some evidence for two other regions of less well defined helical structure. From a preliminary analysis of long-range NOE it is shown that in the E. halophila cytochrome c-551 the general cytochrome c fold is well conserved, including the three conserved helices (residues 2-8, 41-50, 63-76), the regions around the heme ligands (Cys14-Ser15-Ser16-Cys17-His18, Met55) and the omega loop (residues 18-28). In addition, three variable segments of the protein are discussed in detail, one of those including a cis-proline, a feature so far unique in the cytochrome c family. Structural alignments of the E. halophila cytochrome c-551 with two other Pseudomonas cytochrome c5 homologs (Azotobacter vinelandii cytochrome c5 and Chlorobium limicola cytochrome c-555) are provided which are based on sequence similarities and secondary structure alignments.

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Cytochrome c553 from sulfate-reducing bacteria is a low-oxidoreduction-potential cytochrome. The primary and tertiary structures show notable differences when compared to mitochondrial cytochromes. Tyr64 replacement in cytochrome c553 provides evidence that this residue is not directly involved in the potential modulation but is mostly implicated in the hydrogen-bond network around the heme. While the different variants obtained did not induce drastic structural modifications, they did affect the stability of the protein. This decrease of stability in acidic and alkaline environments was observed by variations in the optical spectra and by mass spectrometry. In addition, the mobility of aromatic side-chain was found to be increased in the mutant proteins as monitored by two-dimensional NMR spectroscopy.

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Senktide is a highly specific and potent analog of neurokinin B, the natural ligand of the tachykinin receptor NK-3. The membrane-bound conformation of senktide, interacting with negatively charged membrane vesicles composed of perdeuterated phosphatidylcholine and phosphatidylglycerol (70:30), has been investigated using two-dimensional transferred nuclear Overhauser effect spectroscopy (TRNOESY). The occurrence of an N-methylated phenylalanine in the peptide's sequence induces a cis-trans-isomerisation of the corresponding peptide bond which is slow on the chemical-shift scale. NMR data indicate a much stronger membrane affinity of the trans isomer, allowing the determination of a highly resolved membrane-bound conformation using distance geometry and energy minimization. The membrane-bound backbone conformation of several residues is found to be close to a left-handed helix, certainly due to the presence of nonnatural residues (succinylated N-terminal, N-methyl-phenylalanine) as well as a glycine. The results are discussed in the context of a possible biological relevance of the membrane-bound conformation, in terms of the affinity and specificity of this neuropeptide.

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Experiments with large unilamellar dioleoylphosphatidylcholine vesicles were carried out in order to study the effect of membrane potential on the fluorescence of Oxonol VI. A partition equilibrium of dye between membrane and water was found to exist with a partition coefficient gamma identical to c lipid/c water of about 19,000 (at zero voltage). In the presence of an inside-positive membrane potential, the negatively charged dye accumulates in the intravesicular aqueous space according to a Nernst equilibrium. This leads to an increased adsorption of dye to the inner lipid monolayer and to a concomitant increase of fluorescence. The fluorescence change can be calibrated as a function of transmembrane voltage by generating a potassium diffusion potential in the presence of valinomycin. The intrinsic fluorescence of the membrane-bound dye is not affected by voltage; the whole influence of voltage on the fluorescence results from voltage-dependent partitioning of the dye between water and membrane. The voltage dependence of the apparent partition coefficient can be quantitatively described by a three-capacitor model in which the dye is assumed to bind to adsorption planes located on the hydrocarbon side of the membrane/solution interface. Oxonol VI was found to be suitable for detecting changes of membrane potential associated with the activity of the (Na+ + K+)-ATPase in reconstituted vesicles. When ATP is added to the external medium, pump molecules with the ATP-binding side facing outward become activated; this results in a translocation of net positive charge towards the vesicle interior. Under this condition, fluorescence changes corresponding to (inside-positive) potentials of up to 150-200 mV are observed. After the build-up of the membrane potential, a quasi-stationary state is reached in which the pump current is compensated by a back-flow of charge through passive conductance pathways.

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