Ca2+/Mg2+ exchange in parvalbumin and other EF-hand proteins. A theoretical study.

A remarkable conformational rearrangement occurs upon Ca2+/Mg2+ exchange in the C-terminal EF-hand site (labelled site EF or EF-4) of parvalbumin, as initially established by X-ray crystallography. Such a conformational rearrangement is characterised as follows: (i) the co-ordination number decreases from seven oxygen atoms in the Ca-loaded form to six oxygen atoms in the Mg-loaded form, the heptaco-ordination of Ca2+ corresponding with a skewed pentagonal bipyramid configuration of the seven oxygen atoms, whereas the hexaco-ordination of Mg2+ corresponds with a regular octahedral configuration of the six oxygen atoms; and (ii) Glu101, at the relative position 12 in the EF-hand loop sequence (labelled "Glu12"), acts as a bidentate ligand in the Ca-loaded form and as a monodentate ligand in the Mg-loaded form. As part of the conformational rearrangement, the chi1 dihedral angle undergoes a gauche(+) to gauche(-) transition upon substitution of Ca2+ by Mg2+, whereas the chi2 angle remains practically unchanged and the chi3 angles in both forms adopt a nearly mirror image relationship. In order to understand the molecular mechanisms underlying such a conformational rearrangement, we undertook a theoretical study using the free energy perturbation (FEP) method, starting from high-resolution crystal structures of the same parvalbumin (pike 4. 10 isoform) differing by the substitution of their two cationic sites EF-3 (or CD) and EF-4 (or EF), i.e. the 1pal structure with EF-3(Ca2+) and EF-4(Ca2+), the 4pal structure with EF-3(Ca2+) and EF-4(Mg2+). When Mg2+ is "alchemically" transformed into Ca2+ within the EF-4 site of 4pal, the conformational rearrangement of Glu12 is correctly predicted by the FEP calculation. When Ca2+ is transformed into Mg2+ within the EF-3 site of 4pal, the FEP calculation predicts the topology of the fully Mg-loaded form for which no crystallographic data is presently available. As expected, Glu62 (at the relative position 12 in EF-3 loop) is predicted to be a monodentate residue within a regular octahedral arrangement of six oxygen atoms around Mg2+. We also investigated the behaviour during Ca2+/Mg2+ exchange of two other typical EF-hand proteins, troponin C (TnC) and calmodulin (CaM), for which no three-dimensional structure of their Mg-loaded forms is available so far. It is also predicted that the EF-3 site of TnC and the EF-1 site of CaM have their invariant Glu12 residues switching from the bidentate to the monodentate configuration when Ca2+ is substituted by Mg2+, with six oxygen atoms being observed in the co-ordination sphere of the alchemically generated Mg2+ cation.

[Gamma function extrapolation of dilution curves for pulmonary blood volume determination in chronic bronchitis (author's transl)]. / Extrapolation par la fonction gamma des courbes de dilution. Application au calcul du volume sanguin pulmonaire dans la bronchite chronique.

The risk of incorporating inapparent recirculation under the extrapolated downslope of dye dilution curves is greater with exponential than gamma function extrapolation since the latter makes use of ascending and early descending limbs of the curve. Extrapolation of a true gamma function is not affected by the level at which extrapolation begins. With curves obtained in eight normal subjects, cardiac output (phi) was comparable by exponential and gamma extrapolation if the latter began between 75 and 55% of the peak concentration (Cmax.). phi was underestimated or overestimated according to whether extrapolation began higher or lower than the above limits. Therefore, experimental curves were not true gamma functions. However, the level at which extrapolation began had little effect on pulmonary mean transit time and pulmonary blood volume (PBV) calculated by use of the double injection-single sampling method. Similar effects on phi of the level at which extrapolation by a gamma function begins were found with 24 chronic bronchitics. PBV calculated with a gamma function extrapolated from 0.75 X Cmax. downwards and with an exponential function averaged approximately 340 ml and did not differ from one another. The data suggest that the low values of PBV in chronic bronchitis are not an artifact due to the method of extrapolation.