Claude Pfister

Fluoroaluminates activate transducin-GDP by mimicking the γ-phosphate of GTP in its binding site

Fluoride activation of the cGMP cascade of vision requires the presence of aluminum, and is shown to be mediated by the binding of one AlF‐4 to the GDP/GTP‐binding subunit of transducin. The presence of GDP in the site is required: AlF4 − is ineffective when the site is empty or when GDPßS is substituted for GDP. This sensitivity to the sulfur of GDPßS suggests that AlF4 − is in contact with the GDP. Striking structural similarities between AlF4 − and PO4 −1 lead us to propose that AlF4 − mimics the role of the γ‐phosphate of GTP.

Fast dynamics of halophilic malate dehydrogenase and BSA measured by neutron scattering under various solvent conditions influencing protein stability.

Protein thermal dynamics was evaluated by neutron scattering for halophilic malate dehydrogenase from Haloarcula marismortui (HmMalDH) and BSA under different solvent conditions. As a measure of thermal stability in each case, loss of secondary structure temperatures were determined by CD. HmMalDH requires molar salt and has different stability behavior in H2O, D2O, and in NaCl and KCl solvents. BSA remains soluble in molar NaCl. The neutron experiments provided values of mean-squared atomic fluctuations at the 0.1 ns time scale. Effective force constants, characterizing the mean resilience of the protein structure, were calculated from the variation of the mean-squared fluctuation with temperature. For HmMalDH, resilience increased progressively with increasing stability, from molar NaCl in H2O, via molar KCl in D2O, to molar NaCl in D2O. Surprisingly, however, the opposite was observed for BSA; its resilience is higher in H2O where it is less stable than in D2O. These results confirmed the complexity of dynamics–stability relationships in different proteins. Softer dynamics for BSA in D2O showed that the higher thermostability is associated with entropic fluctuations. In the halophilic protein, higher stability is associated with increased resilience showing the dominance of enthalpic terms arising from bonded interactions. From previous data, it is suggested that these are associated with hydrated ion binding stabilizing the protein in the high-salt solvent.

Guanine nucleotides and magnesium dependence of the association states of the subunits of transducin

When GTPγS is bound to transducin (T), the two subunits Tα·GTPγS and Tβγ dissociate, independently of the ionic environment. When GDP is bound, these subunits are associated as a monomeric Tα·GDP‐Tβγ complex of 75 kDa when the ionic environment is comparable to that of the cytoplasm, but they dissociate in the presence of 10–100 mM Mg2+ or Ca2+ Using this property, the subunits could be separated and purified by a rapid one‐step procedure on an ion‐exchange column (FPLC), and their molecular masses were verified by neutron small angle scattering. The physiological relevances of the dissociating effect of Mg2+ are discussed.

The retinal phototransduction process: enzymatic cascade and regulation

Among cellular systems performing the transduction of an external stimulus, phototransduction in vertebrate rod cells is a unique case which allows convergent approaches to electrophysiological, biophysical and biochemical analyses. The framework of the molecular processes involved in the corresponding enzymatic cascade is now elucidated and can be considered as a model for G protein mediated transductions. We present here the main features of this cascade, its amplification and regulation properties. The mode of stimulation by the aluminofluoride ion is particularly addressed.

Electrical response of beef-heart submitochondrial particles bound to phospholipid-impregnated millipore filters during ATP hydrolysis

1. Beef heart submitochondrial particles bound to asolectin impregnated Millipore filter, according to the method described earlier (Drachev et al. (1974) Nature 249, 321--324), are able to generate, upon addition of ATP, an electrical potential which can be directly measured. 2. The transmembrane electrical potential generated by ATP hydrolysis reaches values up to 80 mV. The half-time required to attain the plateau of potential is paradoxically long (5 to 10 min at room temperature) and is temperature-dependent. Among different phospholipid species which have been used to impregnate the Millipore filter, phosphatidylethanolamine was found to be the most effective for generation of electrical potential. 3. The potential generated by ATP hydrolysis is inhibited by inhibitors of mitochondrial ATPase, by the uncoupler FCCP and by reagents collapsing the membrane potential. 4. Addition of inhibitors of mitochondrial ATPase, when the plateau of potential is attained, results in a decay of potential. This decay of potential is as slow as the generation of potential induced by ATP hydrolysis. 5. The initial rise in electrical potential is proportional to the ATPase activity.

Fast Termination and Adaptation Processes in the cGMP Cascade of Visual Transduction

The main features of the visual phototransduction process, through which light initiates changes of ionic conductance in the retinal rod cell membrane, are now elucidated: cGMP is the soluble transmitter molecule, between the intracellular disk membrane where the light catching pigments are embedded, and the cell membrane which contains the ionic channels. Illumination of rhodopsin results in a fast and intense activation of a cGMP phosphodiesterase. cGMP controles directly the conductance of the cell membrane channels to which it binds. No cyclic nucleotide dependent kinase is involved. This is now the best documented example of a transduction mechanism between a membrane receptor protein (rhodopsin) and membrane bound effectors of cyclic nucleotides (cGMP phosphodiesterase), mediated by a special type of G protein, transducin (see 1 and 2 for recent reviews). It is the archetype of G protein mediated transduction.