Binding of galactose and lactose to ricin. Equilibrium studies.

The interaction of ricin, one of the two lectins of Ricinus sanguineus, with its specific ligands galactose and lactose (4-O-beta-D-galactopyranosyl-D-glucopyranose) has been studied by means of equilibrium dialysis, analytical ultracentrifugation and fluorescence polarization. In the studied concentration range, only one molecule of galactose is bound per molecule of ricin with an association constant, Ka = 6900 m-1 at 4 degrees C. Scatchard plots of equilibrium dialysis data show that two molecules of lactose bind to one molecule of ricin, without modification of molecular weight of the lectin. Together with results of microcalorimetric experiments and agglutination of erythrocytes by ricin, equilibrium dialysis data indicate that the lectin contains two distinct saccharide binding sites. Regardless of the existence of extended sites, it is not possible to select between the two models: (a) two independent sites (Ka1 = 35 000 M-1, Ka2 = 2800 M-1 at 4 degrees C) or (b) two identical sites with negative cooperativity.

[Interaction between ricin hemagglutinin and its ligands, galactose and lactose. Microcalorimetry and equilibrium dialysis]. / Interaction entre l'hémagglutinine de ricin et ses ligands, galactose et lactose. Etude par microcalorimétrie et dialyse à l'équilibre.

The interaction of Ricinus communis hemagglutinin with galactose and lactose has been studied by means of microcalorimetry, equilibrium dialysis and analytical ultracentrifugation. A first class of beta-galactoside-binding sites involves two similar and independent sites of which affinity constants are 2600 M-1 for galactose and 26700 M-1 for lactose at 25 degrees C. The binding of one galactose or one lactose molecule leads to enthalpy changes of--12.3 Kcal and--11 Kcal, respectively. Considering the negative entropy changes of the association, and as for ricin, the binding of galactosides with hemagglutinin is driven by favorable enthalpic contributions. In presence of high lactose concentrations, a second endothermic step of the calorimetric titration curve was observed. This result and the biphasic nature of Scatchard plots of equilibrium dialysis suggest the existence of a second class of binding sites on the lectin molecule. As for ricin, the interaction between these secondary sites and lactose would be entropically driven.

Resolved fluorescence of the two tryptophan residues in horse apomyoglobin.

The composite fluorescence emission from the two tryptophans (W7 and W14) of horse heart apomyoglobin was explored by fluorescence quenching experiments. The fluorescence of the W7 residue is the only one involved in the quenching by iodide or trichloroethanol (TCE) titration. The fluorescence contribution of W7 is 49% of the total apomyoglobin emission, and its spectrum is red-shifted compared to the W14 emission. The fluorescence decay of Trp residues gives an average fluorescence lifetime of 2.06 ns for W14 and 2.84 ns for W7. The static fluorescence quenching by TCE was used to monitor the individual motions of the two tryptophans in apomyoglobin. The short correlation time of W7 (rho = 3 ns) explains why this residue can experience various environments without having to assume the existence of several protein conformations occurring during its lifetime emission.

Implication of the alpha 1 beta 1 interface in the hemoglobin affinity changes. A comparative study between normal and San Diego fully ligated hemoglobins.

The alpha 1 beta 1 interface of normal and mutated San Diego hemoglobins in their fully liganded form was investigated, through the SH vibrational absorption of beta-112 cysteine, by Fourier-transform infrared spectroscopy. The center frequency of this thiol group was significantly shifted in San Diego hemoglobin compared with normal human hemoglobin. Different dimer organization between the two proteins was also revealed by circular dichroism of the heme. These findings agree well with assessment that the alpha 1 beta 1 interface, far from being inert, is involved in the affinity changes of the hemoglobin molecule.

Dynamic and structural properties of glucose oxidase enzyme.

The catalytic oxidation of beta-D-glucose by the enzyme glucose oxidase involves a redox change of the flavin coenzyme. The structure and the dynamics of the two extreme glucose oxidase forms were studied by using infrared absorption spectroscopy of the amide I'band, tryptophan fluorescence quenching and hydrogen isotopic exchange. The conversion of FAD to FADH2 does not change the amount of alpha-helix present in the protein outer shell, but reorganizes a fraction of random coil to beta-sheet structure. The dynamics of the protein interior vary with the redox states of the flavin without affecting the motions of the structural elements near the protein surface. From the structure of glucose oxidase given by X-ray crystallography, these results suggest that the dynamics of the interface between the two monomers are involved in the catalytic mechanism.

Heterogeneous motions within human apohemoglobin.

Conservation of the secondary and tertiary protein organization of human apohemoglobin was observed at temperatures ranging from 7 to 25 degrees C using CD spectra in the far-UV (200-250 nm) and near-UV (250-300 nm) regions. The dynamics of apohemoglobin were probed using fluorescence quenching experiments on the Trp residues and an extrinsic dye (ANS or bis-ANS) located in the heme cavities. The long decay time of the dye emission (> 10 ns) reveals the dynamics of the protein matrix averaged over the whole molecule. The short decay time of the Trp residue emission (congruent with 3 ns) probes the dynamics of their close vicinities. When the temperature rises from 10 to 20 degrees C, the average intraproteic motions throughout the whole apohemoglobin matrix are greatly accelerated, whereas the hydrophobic protein regions around the alpha14, beta15 and beta37 Trp residues appear much less animated. These dynamic differences between the behavior of the softer matrix and the packed rigid regions containing the tryptophans could be one of the requisites for apohemoglobin stability. We suspect that the highly rigid tryptophan domains in human apohemoglobin are likely to be knots.

Solvent-induced structural distortions of horse metmyoglobin.

Structural and dynamic constraints produced by the surrounding solvent on the aquometmyoglobin molecule were investigated by means of circular dichroism and Fourier-transform infrared spectroscopies, tritium/hydrogen exchange kinetics and small-angle neutron-scattering experiments. Formamide and ethanol were chosen as cosolvents because they are known to increase and decrease protein activity, respectively. The CD measurements in the Soret region show that no changes occur in the heme molecular structure nor in the protein near the heme. The results of proton-exchange kinetics experiments indicate that the conformational dynamics of aquometmyoglobin is only marginally affected by the cosolvents. However, the small-angle neutron-scattering spectra strongly suggest that these cosolvents induce some distortions of the tertiary conformation. According to the ultraviolet CD and Fourier-transform infrared data, the alteration of the tertiary conformation results from changes in both the number of intrachain hydrogen bonds and the structures of beta turns of type I' for formamide and of type II for either of the two cosolvents. The use of several techniques allows the present approach to demonstrates that the myoglobin structure is extremely sensitive to its environmental conditions.

Alteration of heme axial ligands in hemoglobin by organic solvents analyzed by CD, FTIR, and XANES techniques.

The effects of mixed solvents on the ligand binding site in hemoglobin have been investigated though three spectroscopic techniques. Two classes of organic solvents (amides and alcohols) known to increase or decrease the hemoglobin affinity have been chosen for this study. The analysis of the iron CO stretching band shows that the ligand binding sites of alpha CO and beta CO subunits inside the alpha 2 beta 2 hemoglobin tetramer exhibit multiple conformations. From the circular dichroism and X-ray absorption near-edge structure data, it appears that no core deformation or heme reorientation occur with the affinity changes. The iron-ligand average bond angle is the sole parameter that depends on the external solvent. Since cosolvents seem to affect the dynamics rather than the hindrance of the heme cavity, we suggest that the protein affinity could be associated with a hierarchy of subtle dynamic states.

Solvent effects on horse apomyoglobin dynamics.

The effects of the solvent conditions (buffer pH 9, 8, or 7 or buffer pH 6.5 alone or mixed with 3.2% ethanol or 6.2% formamide) on the protein dynamics of horse apomyoglobin were investigated through tryptophan fluorescence quenching, spectra, and decay properties. Raising the pH (which induces discontinuous protein conformation changes) increases the structural fluctuations inside the hydrophobic A, G, and H helix core. Mixed solutions containing either 3.2% ethanol or 6.2% formamide (which redistribute water molecules on the protein surface) produce protein dynamics changes in the vicinity of the two Trp residues, without inducing particular constraints on these very residues. Formamide increases, in the same way, the polarity and the protein flexibility while ethanol reduces both. The present fluorescence work also shows that, whatever the outside solvent, the two Trp residues W7 and W14, embedded in the A, G, and H helix core, are equally and statistically reached by small molecules diffusing inside the protein matrix. Hydrogen-tritium exchange measurements on the protein in mixed solvents reveal that the dynamics of the A, G, and H helix cluster and of the B and E helixes are greatly influenced by the nature of the outside medium. A small amount of formamide in the buffer increases the protein fluctuations while an ethanol-water mixture reduces them. We suggest that the hydratation state of the protein surface could be the relevant parameter of the protein dynamics.