Purification, crystallization and preliminary X-ray crystallographic analysis of the receiver and stalk domains (PA3346RS) of the response regulator PA3346 fromPseudomonas aeruginosaPAO1

Abstract

The regulatory domain (PA3346RS), comprising the receiver and stalk domains, of the response regulator PA3346 requires phosphorylation for activation with magnesium ions as cofactors in order to modulate the downstream protein phosphatase activity for the regulation of swarming motility in Pseudomonas aeruginosa PAO1. Fusion‐tagged recombinant PA3346RS of total molecular mass 25.3 kDa has been overexpressed in Escherichia coli, purified using Ni2+–NTA and Q‐Sepharose ion‐exchange columns and crystallized using the hanging‐drop vapour‐diffusion method. X‐ray diffraction data were collected from PA3346RS crystals to 2.0 Å resolution. The crystal belonged to space group P41 or P43, with unit‐cell parameters a = 82.38, c = 73.34 Å. Preliminary analysis indicated the presence of a dimer of PA3346RS in the asymmetric unit, with a solvent content of 48.6%.

Impact Analysis of Composite Laminate Shell Structures

“…The contact forces with impactor velocities of 38.1 m/s, 25.4 m/s and 12.7 m/s are plotted in Figure 4. As shown form the figure, the numerical results of the contact force obtained by the present method are in a good agreement with the results from Wu & Chang [10].…”

“…Substituting eqs(1), (3), ( 4), (10) into eq (9), leads to a integro-differential equation in terms of the dependent variable of indension s as follows. For the loading period…”

“…T300/934 graphite epoxy composite with the stacking sequence of [ ] S 2 90 / 45 / 45 / 0 − was used for the study. The material properties of T300/934 are listed in Table 1 (Wu & Chang [10]). The length, width and thickness of the composite plate are 76.2mm, 76.2mm and 2.54mm, respectively.…”

Purification, crystallization and preliminary X-ray crystallographic analysis of the phosphatase domain (PA3346PD) of the response regulator PA3346 fromPseudomonas aeruginosaPAO1

“…Certain bacterial pathogens, including P. aeruginosa, possess 'hybrid'-type TCSs, where the phosphoryl group is transferred from an HK to a histidine-containing phosphotransfer (Hpt) protein and subsequently to the downstream cytoplasmic RR, as shown in Fig. 1 (Wu et al, 2011).…”

“…After the N-terminal REC domain of PA3346 receives a stimulus, the phosphatase activity of PA3346 is increased to dephosphorylate the anti-anti-factor PA3347 to regulate the downstream genes ( Fig. 1; Wu et al, 2011;Bhuwan et al, 2012). This signal pathway has been demonstrated to be involved in flagellar biosynthesis and the chemotaxis response that plays an essential role in its virulence (Feldman et al, 1998;Bhuwan et al, 2012).…”