Impact of A90P, F106L and H64V mutations on neuroglobin stability and ligand binding kinetics.

Human neuroglobin (Ngb) is a hexacoordinated globin which binds some small ligands. Its function is still not well-established, even though Ngb seems to be implicated in the protection against neurodegenerative diseases. It has been shown by molecular dynamics and crystallography that ligand binding could occur thanks to a haem sliding mechanism specific to Ngb. In this paper, we studied some regions which could participate in this mechanism. We used UV-visible spectroscopy, CD and NMR to have a look on the protein structure and NMR and stopped-flow to study the ligand binding properties of the proteins. In the haem environment we mutated the distal histidine H64, the alanine A90 which is on the proximal F helix and the phenylalanine F106 which is close to the haem. We showed that both H64V and A90P variants, which affect the haem coordination, seemed to be important to haem and protein secondary structure stabilities whereas F106L mutation did not affect those properties. Then we confirmed that the cyanide binding kinetics were isomer dependent on wild-type Ngb and A90P and F106L variants. H64V Ngb variant had a behavior similar to wild-type Mb or Hb with a loss of the haem kinetic differentiation. Moreover, our results suggested that one haem isomer was more sensitive to A90P and F106L mutations. Those results brought some evidence that the haem sliding mechanism could occur for the cyanide binding and could be haem isomer dependent. The isomer forms may play distinct roles for the potential function of Ngb in vivo.

Isotopic double-labeling of two honeybee odorant-binding proteins secreted by the methylotrophic yeast Pichia pastoris.

Odorant-binding proteins (OBPs) are soluble, low-molecular-weight proteins secreted in the sensillum lymph surrounding the dendrites of olfactory sensilla from a wide range of insect species. These proteins play a role in the solubilization, transport and/or deactivation of pheromones and odorants. In order to study the relationships between the molecular structure in solution and their ligand-binding properties, we have (13)C/(15)N-double-labeled two divergent honeybee OBPs, called ASP1 and ASP2, in sufficient quantities to permit a full determination of the structure and dynamics using heteronuclear NMR spectroscopy. The recombinant labeled proteins produced by the methylotrophic yeast Pichia pastoris have been secreted into a buffered minimal medium using native insect signal peptide. Mass spectrometry and Edman sequencing showed a native-like processing with a labeling efficiency of secreted proteins greater than 98%. After dialysis, the recombinant proteins were purified to homogeneity by one-step reversed-phase liquid chromatography. The final yield after 4-day shake-flask liquid culture was approximately 60 and 100 mg/L for ASP1 and ASP2, respectively. The inexpensive overproduction of labeled recombinant ASP1 and ASP2 should allow NMR studies of the structures and ligand-binding analysis in order to understand the relationships between structure and biological function of these proteins.