Involvement of the distal Arg residue in Cl⁻ binding of midge larval haemoglobin.

Monomeric haemoglobin component V (Hb V) from the larva of the midge Propsilocerus akamusi shows high Cl⁻ affinity under high salt concentrations at acidic pH. In order to understand the structural changes that depend on Cl⁻ binding, crystal structures of Hb V were determined under acidic high-salt conditions and the structural changes arising from different haem-bound ligands were simulated. Crystal structures of Hb V under acidic high-salt conditions indicated that the side chain of ArgE10 on the distal face of the haem contributes to stabilizing haem-bound Cl⁻. The conformation of the Arg side chain in the Cl⁻-bound form was almost identical to that in ligated Hb V at neutral pH but not to that in met Hb V under acidic salt-free conditions. Furthermore, preliminary molecular-dynamics simulations also indicated that the swinging of the Arg side chain into the haem pocket depends on Cl⁻ ligation. This result suggests that, like pH change, Cl⁻ binding affects the location of the distal Arg residue. Owing to the increased positive electrostatic potential observed in the haem pocket at acidic pH, it was concluded that electrostatic changes caused by pH change and anionic ligand binding may affect the behaviour of the polar Arg residue.

Side-necked turtle (Pleurodira, Chelonia, reptilia) hemoglobin: cDNA-derived primary structures and X-ray crystal structures of Hb A.

Red blood cells of yellow-spotted river turtles (Podocnemis unifilis, Pleurodira, Chelonia, REPTILIA) have two hemoglobin (Hb) components, Hb A and Hb D. We purified the hemoglobin component homologous to amniote (reptiles, birds, and mammals) adult Hb A which comprises two identical α(A) -globin polypeptides and two identical ß-globin polypeptides. To establish the crystal structure of Podocnemis Hb A, we first determined the globin primary structures using cDNA nucleotide sequencing with the assistance of protein sequencing. The purified Podocnemis Hb A produced a different form of crystal for each of the two different buffer systems used: form A, tetragonal crystals (space group, P41212), produced under neutral pH (pH 7-8) conditions; and form B, hexagonal crystals (space group, P6122), produced under high alkaline pH (pH 11-13) conditions. Single crystals of the two forms were examined by Raman microscopy with an excitation of 532 nm, indicating their structural differences. The crystal structures of the two forms were constructed by X-ray crystallographic diffraction at a resolution of 2.20 Å for form A and 2.35 Å for form B. The differences of the tertiary and quaternary structures of the two forms were marginal; however, one clear difference was found in helix structure. When comparing Podocnemis Hb A with Hb A from specimens in other taxa, such as Anser indicus (birds) and Homo sapiens (mammals) by SHELXPRO, the root mean square deviation (RMSD) between the corresponding Cα atoms of the two globins does not exceed 2.0 Å. These low values indicate the crystal structures resemble each other. Our data on X-ray crystal structures and Raman spectra not only reveal the first findings on the two crystal forms of Podocnemis unifilis Hb A but also provide the first refined models for reptilian adult Hb A.

pH-dependent structural changes in haemoglobin component V from the midge larva Propsilocerus akamusi (Orthocladiinae, Diptera).

Haemoglobin component V (Hb V) from the midge larva Propsilocerus akamusi exhibits oxygen affinity despite the replacement of HisE7 and a pH-dependence of its functional properties. In order to understand the contribution of the distal residue to the ligand-binding properties and the pH-dependent structural changes in this insect Hb, the crystal structure of Hb V was determined under five different pH conditions. Structural comparisons of these Hb structures indicated that at neutral pH ArgE10 contributes to the stabilization of the haem-bound ligand molecule as a functional substitute for the nonpolar E7 residue. However, ArgE10 does not contribute to stabilization at acidic and alkaline pH because of the swinging movement of the Arg side chain under these conditions. This pH-dependent behaviour of Arg results in significant differences in the hydrogen-bond network on the distal side of the haem in the Hb V structures at different pH values. Furthermore, the change in pH results in a partial movement of the F helix, considering that coupled movements of ArgE10 and the F helix determine the haem location at each pH. These results suggested that Hb V retains its functional properties by adapting to the structural changes caused by amino-acid replacements.

Crystal structures of two hemoglobin components from the midge larva Propsilocerus akamusi (Orthocladiinae, Diptera).

The polymorphic components of hemoglobin (Hb) of the midge larva Propsilocerus akamusi were classified into two distinct types dependent on their spectroscopic properties, normal absorption (N) and low absorption (L). Analyses of the amino acid sequences of component VII (N-type Hb) and component V (L-type Hb) from P. akamusi indicated that one remarkable difference is the replacement of the distal histidine (His) with isoleucine (Ile) in component V. To clarify the structural differences between the two Hb components, we determined the crystal structures of components V and VII at resolutions of 1.64 A and 1.50 A, respectively. These crystal structures indicated a short additional helix comprising three amino acid residues at the C-terminal region in component V, and a typical globin fold including eight helices in component VII. Comparison of the heme regions of the Hb components suggests that the structural changes of the heme region in component V on ligation differ from that of usual Hb.

Thermal ablation of FEL irradiation using gypsum as an indicator.

Thermal effects produced in a laser-irradiated sample were studied by micro-X-ray diffraction and micro-Fourier transform infrared spectroscopy (FTIR). Gypsum, transformed into bassanite at 124 degrees C and into anhydrite at 147 degrees C, was used as a thermal indicator. Pit formation by a wavelength-tunable free electron laser (FEL) irradiation on the gypsum pellet maximized at a wavelength of 3.0 microm, 2 mJ/shot, and pits were not detected in those irradiated at 2.6 or 3.8 microm compared with the maximum at 3.0 microm and diminished at 2.0 or 4.0 microm in the human tooth case. Micro-X-ray diffraction and micro-FTIR did not reveal any appreciable bassanite or anhydrite in the irradiated regions. From the laser ablation viewpoint, these results allow the FEL ablation to be considered as plasma or evaporative ones. This study indicated that the micro-pulse of laser was effective to prevent thermal damages of laser irradiation.

Crystallization and preliminary X-ray diffraction study of hemoglobin D from the Aldabra giant tortoise, Geochelone gigantea.

Hemoglobin D (Hb D) from the Aldabra giant tortoise, Geochelone gigantea, was crystallized by the hanging drop vapor diffusion technique with a precipitant solution containing 10% polyethylene glycol 3350 and 50 mM HEPES-Na, pH 7.5. The Hb D crystals of G. gigantea, which diffract to at least a 2.0 A resolution, belong to the monoclinic space group C2 with unit cell dimensions of a = 112.1 A, b = 62.4 A, c = 54.0 A, and beta = 110.3 degrees. One alphabeta dimer molecule of Hb D existed in an asymmetric unit, with a calculated value of Vm of 2.77 A(3)Da(-1).