Crystallization and structure determination of bovine profilin at 2.0 A resolution

Profilin regulates the behavior of the eukaryotic microfilament system through its interaction with non-filamentous actin. It also binds several ligands, including poly(L-proline) and the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). Bovine profilin crystals (space group C2; a = 69.15 A, b = 34.59 A, c = 52.49 A; alpha = gamma = 90 degrees, beta = 92.56 degrees) were grown from a mixture of poly(ethylene glycol) 400 and ammonium sulfate. X-ray diffraction data were collected on an imaging plate scanner at the DORIS storage ring (DESY, Hamburg), and were phased by molecular replacement, using a search model derived from the 2.55 A structure of profilin complexed to beta-actin. The refined model of bovine profilin has a crystallographic R-factor of 16.5% in the resolution range 6.0 to 2.0 A and includes 128 water molecules, several of which form hydrogen bonds to stabilize unconventional turns. The structure of free bovine profilin is similar to that of bovine profilin complexed to beta-actin, and C alpha atoms from the two structures superimpose with an r.m.s. deviation of 1.25 A. This value is reduced to 0.51 A by omitting Ala1 and the N-terminal acetyl group, which lie at a profilin-actin interface in crystals of the complex. These residues display a strained conformation in crystalline profilin-actin but may allow the formation of a hydrogen bond between the N-acetyl carbonyl group of profilin and the phenol hydroxyl group of Tyr188 in actin. Several other actin-binding residues of profilin show different side-chain rotomer conformations in the two structures. The polypeptide fold of bovine profilin is generally similar to those observed by NMR for profilin from other sources, although the N terminus of Acanthamoeba profilin isoform I lies in a distorted helix and the C-terminal helix is less tilted with respect to the strands in the central beta-pleated sheet than is observed in bovine profilin. The majority of the aromatic residues in profilin are exposed to solvent and lie in either of two hydrophobic patches, neither of which takes part in an interface with actin. One of these patches is required for binding poly(L-proline) and contains an aromatic cluster comprising the highly conserved residues Trp3, Tyr6, Trp31 and Tyr139. In forming this cluster, Trp31 adopts a sterically strained rotamer conformation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Internal cleavage of the inhibitory 7B2 carboxyl-terminal peptide by PC2: a potential mechanism for its inactivation

The neuroendocrine protein 7B2 contains two domains, a 21-kDa protein required for prohormone convertase 2 (PC2) maturation and a carboxyl-terminal (CT) peptide that inhibits PC2 at nanomolar concentrations. To determine how the inhibition of PC2 is terminated, we studied the metabolic fate of the 7B2 CT peptide in RinPE-7B2, AtT-20/PC2-7B2, and alphaTC1-6 cells. Extracts obtained from cells labeled for 6 h with [3H]valine were subjected to immunoprecipitation using an antibody raised against the extreme carboxyl terminus of r7B2, and immunoprecipitated peptides were separated by gel filtration. All three cell lines yielded two distinct peaks at about 3.5 kDa and 1.5 kDa, corresponding to the CT peptide and a smaller fragment consistent with cleavage at an interior Lys-Lys site. These results were corroborated using a newly developed RIA against the carboxyl terminus of the CT peptide which showed that the intact CT peptide represented only about half of the stored CT peptide immunoreactivity, with the remainder present as the 1.5-kDa peptide. Both peptides could be released upon phorbol 12-myristate 13-acetate stimulation. We investigated the possibility that PC2 itself could be responsible for this cleavage by performing in vitro experiments. When 125I-labeled CT peptide was incubated with purified recombinant PC2, a smaller peptide was generated. Analysis of CT peptide derivatives for their inhibitory potency revealed that CT peptide 1-18 (containing Lys-Lys at the carboxyl terminus) represented a potent inhibitor, but that peptide 1-16 was inactive. Inclusion of carboxypeptidase E (CPE) in the reaction greatly diminished the inhibitory potency of the CT peptide against PC2, in line with the notion that the CT peptide cleavage product is not inhibitory after the removal of terminal lysines by CPE. In summary, our data support the idea that PC2 cleaves the 7B2 CT peptide at its internal Lys-Lys site within secretory granules; deactivation of the cleavage product is then accomplished by CPE, thus providing an efficient mechanism for intracellular inactivation of the CT peptide.     

A method for the identification and quantitative investigation of denatured proteins in mixtures based on computer comparison of amino-acid patterns

Summary A procedure is described in which stepwise regression is adapted to permit comparison of the amino-acid pattern from a composite sample with those of simple substances arranged in an easily accessible data bank. The computer program automatically selects from the bank those proteins whose amino-acid patterns best correspond to that of the sample, and calculates the proportion of the proteins contained in the mixture.The procedure is applicable to food analytical problems that involve the identification and determination of proteins in mixtures, and also to situations in which the properties of the proteins have been altered by denaturation or enzymatic degradation. The determination is limited to 3–4 proteins in the mixture.

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Identifizierung und quantitative Bestimmung einzelner Proteine in Mischungen von denaturierten Proteinen mit Hilfe ihrer Aminosäauremuster

Zusammenfassung Mit Hilfe einer stufenweisen Regressionsberechnung konnen Aminosäu-renmuster eines zusammengesetzten Proteins mit einfachen Proteinen verglichen werden. Mit dieser Berechnung werden aus einer Sammlung von Vergleichsproteinen automatisch solche Proteine ausgewählt, deren Aminosäurenmuster am besten zu der der Probe paßt. Gleichzeitig können dadurch die Verhältnisse der verschiedenen Proteine in der Mischung berechnet werden. Durch these Arbeitsweise konnen weiterhin bei nahrungsanalytischen Problemen Proteine in Mischungen identifiziert and quantitativ erfaßt werden, selbst worn die Proteine denaturiert oder enzymatisch abgebaut sind, jedoch ist das Verfahren auf 3–4 Proteine in der Mischung begrenzt.

     

The electrical conductivities of poly(3.6-pyridazine) and poly(3.6-pyridazine sulphide)

Summary We have chemically synthesized poly(3.6-pyridazine),(PPd), and poly(3.6-pyridazine sulphide), (PPdS), and studied the electrical conductivities of these polymers.     

A bandwidth enhancement technique for mobile handset antennas using wavetraps

A novel technique for enhancing the impedance bandwidth of wireless terminal antennas is presented. By introducing resonant short circuit transmission lines to the long sides of the chassis edges, an effective electrical shortening of the terminal ground plane is achieved. This effect has been used to realize terminal ground planes with resonant lengths at high frequencies, such as GSM 1800/1900 MHz or UMTS 2 GHz, thereby extending the impedance bandwidth. The proposed technique has been validated by simulations and measurements. Three typical applications are presented where the introduction of wavetraps improves the bandwidth and/or in-band performance.     

Evaluation of nitrogen dioxide scavengers during delivery of inhaled nitric oxide

Tissue hypoxia is an important cause for the development of multisystem organ failure in the critically ill. Achieving adequate haemodynamic support of oxygen demand is the mainstay of treatment in these patients. Controversies regarding therapeutic end-points do exist but in general maintaining oxygen delivery by ensuring adequate cardiac output, oxygen saturation and haemoglobin is important in the critically ill.