Crystallization and preliminary X-ray investigation of calmodulin

Crystals of rat testis calmodulin, a multifunctional Ca2+-binding protein have been grown from solutions of 2-methyl-2,4-pentanediol. The crystals are triclinic, space group P1, with a = 29.79(4) A, b = 53.74(7) A, c = 24.78(3) A, alpha = 93.46(2)degrees, beta = 96.98(2)degrees, and gamma = 89.05(3)degrees. There is 1 calmodulin molecule per unit cell. The crystals are quite stable to x-rays and diffract beyond 2.5 A resolution.     

Gender-related biases in admission decisions

This study tested the effects of applicant gender and attractiveness on admission ratings, personality attributions and causal attributions in the student admission decision-making process in allied health education. The study showed that although gender stereotypes are very strong, they did not affect admission ratings. However, the study did reveal that attractive candidates received higher admission ratings than unattractive applicants and the past successes of attractive applicants were more likely attributed to internal causes while the past successes of unattractive applicants were more often attributed to external causes.     

The motion of bubbles in a sinusoidally oscillating liquid in microgravity

The sinusoidal motion of single, spherical bubbles in microgravity was studied experimentally aboard the U.S. Space Shuttle. Tests were performed to determine the effect of frequency, acceleration amplitude, bubble size, and fluid viscosity on bubble motion. Five test cells each containing a single bubble were subjected to rectilinear, sinusoidal oscillations. Three nominal bubble sizes and three liquids were used to cover a range of Stokes numbers from 1.3 to 21 and Reynolds numbers from 0.6 to 75. Bubble motion was recorded by video. The ratio of bubble motion amplitude to container motion amplitude was found to be essentially independent of the actual container motion amplitude. Therefore, this ratio could be plotted against frequency to obtain a frequency response for each case. This ratio was found to rise sharply from zero at zero frequency and then approach an asymptote at high frequencies. The strong effect of the walls in these experiments caused the amplitude of bubble motion to be reduced somewhat from that expected for an infinite fluid.     

Crystallization of the EGF receptor ectodomain on US space mission STS-47

Although biochemists working in the field of biological signal transduction have characterized cell surface receptors for numerous growth factors within the past ten years, none of the three-dimensional structures could be obtained for these important proteins which represent major components of the cells' growth control system. Now, the extracellular ligand binding domain of the EGF receptor was crystallized in the presence of EGF under microgravity on US Shuttle mission STS-47. In 8 out of 9 experiments prepared under different conditions crystal growth was observed. One of these space-grown crystals showed higher diffraction quality than all crystals previously obtained in the laboratory. It allowed, for the first time, evaluation of the real space group by partial data collection.     

Identification of a Novel Inhibition Site in Translocase MraY Based upon the Site of Interaction with Lysis Protein E from Bacteriophage ϕX174

Translocase MraY is the site of action of lysis protein E from bacteriophage ?X174. Previous genetic studies have shown that mutation F288L in transmembrane helix 9 of E. coli MraY confers resistance to protein E. Construction of a helical wheel model for transmembrane helix 9 of MraY and the transmembrane domain of protein E enabled the identification of an Arg‐Trp‐x‐x‐Trp (RWxxW) motif in protein E that might interact with Phe288 of MraY and the neighbouring Glu287. This motif is also found in a number of cationic antimicrobial peptide sequences. Synthetic dipeptides and pentapeptides based on the RWxxW consensus sequence showed inhibition of particulate E. coli MraY activity (IC₅₀ 200–600 μM ), and demonstrated antimicrobial activity against E. coli (MIC 31–125 μg mL^?1). Cationic antimicrobial peptides at a concentration of 100 μg mL^?1 containing Arg‐Trp sequences also showed 30–60 % inhibition of E. coli MraY activity. Assay of the synthetic peptide inhibitors against recombinant MraY enzymes from Bacillus subtilis, Pseudomonas aeruginosa, and Micrococcus flavus (all of which lack Phe288) showed reduced levels of enzyme inhibition, and assay against recombinant E. coli MraY F288L and an E287A mutant demonstrated either reduced or no detectable enzyme inhibition, thus indicating that these peptides interact at this site. The MIC of Arg‐Trp‐octyl ester against E. coli was increased eightfold by overexpression of mraY, and was further increased by overexpression of the mraY mutant F288L, also consistent with inhibition at the RWxxW site. As this site is on the exterior face of the cytoplasmic membrane, it constitutes a potential new site for antimicrobial action, and provides a new cellular target for cationic antimicrobial peptides.     

Catalytic promiscuity in the alpha/beta-hydrolase superfamily: hydroxamic acid formation, C--C bond formation, ester and thioester hydrolysis in the C--C hydrolase family

The haloperoxidase family of alpha/beta-hydrolases contains enzymes of several different catalytic activities, including esterases, C--C hydrolases and cofactor-independent haloperoxidases (perhydrolases), but the molecular basis of this catalytic promiscuity is not fully understood. The C--C hydrolase enzyme MhpC from E. coli is shown to possess esterase and thioesterase activity, and the ability to activate hydroxylamine as a nucleophile to form hydroxamic acid products. The ratio of these activities was examined for nine site-directed mutant enzymes that contained mutations at nonessential residues in the enzyme active site. Higher levels of esterase and thioesterase activity were found in mutants Phe173Gly and Trp264Gly; this might be due to increased amounts of space in the active site. Higher levels of hydroxamic acid formation activity were found in mutant Asn109His-a mutation found in many haloperoxidase enzymes. Wild-type and mutant MhpC enzymes were also capable of C--C bond formation in organic solvents, and the highest activity was observed in nonpolar solvents. The results provide experimental support for the catalytic promiscuity shown in this family of enzymes, and indicate that differences in catalytic function can be introduced by point mutations.     

Substrate selectivity and biochemical properties of 4-hydroxy-2-keto-pentanoic acid aldolase from Escherichia coli

4-Hydroxy-2-keto-pentanoic acid aldolase from Escherichia coli was identified as a class I aldolase. The enzyme was found to be highly selective for the acetaldehyde acceptor but would accept alpha-ketobutyric acid or phenylpyruvic acid in place of the pyruvic acid carbonyl donor.     

Directed evolution of a non-heme-iron-dependent extradiol catechol dioxygenase: identification of mutants with intradiol oxidative cleavage activity

The non-heme-iron(II)-dependent extradiol catechol dioxygenases catalyse the oxidative cleavage of substituted catechols found on bacterial aromatic degradation pathways. The reaction mechanism of the extradiol dioxygenases is believed to proceed through the same proximal hydroperoxide intermediate as the iron(III)-dependent intradiol catechol dioxygenases. Directed evolution was carried out on members of the class III extradiol catechol dioxygenases, by using 1) error-prone polymerase chain reaction, 2) a primer-based cross-over method; the mutant dioxygenases were then screened for their ability to process a range of substituted catechols. Several mutant enzymes were found to show higher activity towards certain substituted catechols, including 4-chlorocatechol, and higher affinity for the iron(II) cofactor. Two mutants isolated from error-prone PCR of Escherichia coli MhpB (mutants R215W and K273R) were found to produce a mixture of extradiol and intradiol cleavage products, as detected by GC-MS and 1H NMR spectroscopy. The residue corresponding to K273 in protocatechuate 4,5-dioxygenase (LigAB), Val244, is located approximately 12 A from the iron(II) centre, but close to the putative dioxygen channel; R215 is found on a sequence loop not present in LigB.     

Expression of TASK and TREK, two-pore domain K+ channels, in human myometrium

Two-pore domain K+ channels are an emerging family of K+ channels that may contribute to setting membrane potential in both electrically excitable and non-excitable cells and, as such, influence cellular function. The human uteroplacental unit contains both excitable (e.g. myometrial) and non-excitable cells, whose function depends upon the activity of K+ channels. We have therefore investigated the expression of two members of this family, TWIK (two-pore domain weak inward rectifying K+ channel)-related acid-sensitive K+ channel (TASK) and TWIK-related K+ channel (TREK) in human myometrium. Using RT-PCR the mRNA expression of TASK and TREK isoforms was examined in myometrial tissue from pregnant women. mRNAs encoding TASK1, 4 and 5 and TREK1 were detected whereas weak or no signals were observed for TASK2, TASK3 and TREK2. Western blotting for TASK1 gave two bands of approximately 44 and 65 kDa, whereas TREK1 gave bands of approximately 59 and 90 kDa in myometrium from pregnant women. TASK1 and TREK1 immunofluorescence was prominent in intracellular and plasmalemmal locations within myometrial cells. Therefore, we conclude that the human myometrium is a site of expression for the two-pore domain K+ channel proteins TASK1 and TREK1.