Haemoglobin Engineering: For fun and money

AbstractThe recent transplantation of an unusual allosteric effect from crocodile to human haemoglobin has implications for both molecular evolution and the engineering of artificial blood substitutes

A novel bacterial l-arginine sensor controlling c-di-GMP levels in Pseudomonas aeruginosa

Nutrients such as amino acids play key roles in shaping the metabolism of microorganisms in natural environments and in host–pathogen interactions. Beyond taking part to cellular metabolism and to protein synthesis, amino acids are also signaling molecules able to influence group behavior in microorganisms, such as biofilm formation. This lifestyle switch involves complex metabolic reprogramming controlled by local variation of the second messenger 3′, 5′-cyclic diguanylic acid (c-di-GMP). The intracellular levels of this dinucleotide are finely tuned by the opposite activity of dedicated diguanylate cyclases (GGDEF signature) and phosphodiesterases (EAL and HD-GYP signatures), which are usually allosterically controlled by a plethora of environmental and metabolic clues. Among the genes putatively involved in controlling c-di-GMP levels in P. aeruginosa, we found that the multidomain transmembrane protein PA0575, bearing the tandem signature GGDEF-EAL, is an l-arginine sensor able to hydrolyse c-di-GMP. Here, we investigate the basis of arginine recognition by integrating bioinformatics, molecular biophysics and microbiology. Although the role of nutrients such as l-arginine in controlling the cellular fate in P. aeruginosa (including biofilm, pathogenicity and virulence) is already well established, we identified the first l-arginine sensor able to link environment sensing, c-di-GMP signaling and biofilm formation in this bacterium

Structure and metal-binding properties of PA4063, a novel player in periplasmic zinc trafficking by Pseudomonas aeruginosa

The capability to obtain essential nutrients in hostile environments is a critical skill for pathogens. Under zinc-deficient conditions, Pseudomonas aeruginosa expresses a pool of metal homeostasis control systems that is complex compared with other Gram-negative bacteria and has only been partially characterized. Here, the structure and zinc-binding properties of the protein PA4063, the first component of the PA4063-PA4066 operon, are described. PA4063 has no homologs in other organisms and is characterized by the presence of two histidine-rich sequences. ITC titration detected two zinc-binding sites with micromolar affinity. Crystallographic characterization, performed both with and without zinc, revealed an α/β-sandwich structure that can be classified as a noncanonical ferredoxin-like fold since it differs in size and topology. The histidine-rich stretches located at the N-terminus and between β3 and β4 are disordered in the apo structure, but a few residues become structured in the presence of zinc, contributing to coordination in one of the two sites. The ability to bind two zinc ions at relatively low affinity, the absence of catalytic cavities and the presence of two histidine-rich loops are properties and structural features which suggest that PA4063 might play a role as a periplasmic zinc chaperone or as a concentration sensor useful for optimizing the response of the pathogen to zinc deficiency

N-oxide sensing in Pseudomonas aeruginosa: expression and preliminary 1188 RINALDO ET AL. characterization of DNR, an FNR-CRP type transcriptional regulator. Biochem Soc Trans 33: 184–186

Abstract In denitrifying bacteria, the concentration of NO is maintained low by a tight control of the expression and activity of nitrite and NO reductases. Regulation involves redox-linked transcription factors, such as those belonging to the CRP-FNR (cAMP receptor protein-fumarate and nitrate reductase regulator) superfamily, which act as oxygen and N-oxide sensors. Given that few members of this superfamily have been characterized in detail, we have cloned, expressed and purified the dissimilative nitrate respiration regulator from Pseudomonas aeruginosa. To gain insights on the structural properties of the dissimilative nitrate respiration regulator, we have also determined the aggregation state of the purified protein and its ability to bind hydrophobic compounds such as 8-anilino-1-naphthalenesulphonic acid

Aplysia limacina myoglobin cDNA cloning: an alternative mechanism of oxygen stabilization as studied by active-site mutagenesis.

The isolation and cloning of the cDNA coding for myoglobin (Mb) from the mollusc Aplysia limacina is reported here. Five amino acid differences from the previously published protein sequence have been found in positions 22, 26, 27, 77 and 80 by back transplanting the cDNA; some of these may be relevant for overall structure stabilization in this Mb. High-level expression of the holoprotein in Escherichia coli has been achieved in the presence of the haem precursor delta-aminolevulinic acid, underlying the importance of tuning haem and apoprotein biosynthesis to achieve high-level expression of haemproteins in bacteria. The recombinant protein is identical to the protein purified from the mollusc buccal muscle. Native A. limacina Mb has an oxygen dissociation rate constant of 70 s(-1) [as compared with the value of 15 s(-1) for sperm whale Mb, which displays His(E7) and Thr(E10)] (amino acid positions are referred to within the eight helices A-H of the globin fold). In order to understand the mechanism of oxygen stabilization in A. limacina Mb, we have prepared and investigated three active-site mutants: two single mutants in which Val(E7) and Arg(E10) have been replaced by His and Thr, respectively, and a double mutant carrying both mutations. When Arg(E10) is substituted with Thr, the oxygen dissociation rate constant is increased from 70 s(-1) to more than 700 s(-1), in complete agreement with the previously proposed role of the former residue in ligand stabilization. In the His(E7)-containing single and double mutants, both displaying high oxygen dissociation rates, the stabilization of bound oxygen by the distal His is insufficient to slow down the ligand dissociation rate constant to the value of sperm whale Mb. These results essentially prove the hypothesis that in A. limacina Mb a mechanism of oxygen stabilization involving Arg(E10), and thus different from that mediated by His(E7), has evolved