Characterization of Mg2+-induced conformational change in the 50S ribosomal subunit by differential hydrogen exchange.

The technique of differential hydrogen exchange allows detection of a conformational change in the 50S subunit of Escherichia coli ribosome when the magnesium concentration is lowered in a range where ribosomal activity is fully preserved. This change is characterized by a seventy-fold acceleration of about thirty labile hydrogens in the case of a Mg2+ jump from 10 mM to 2 mM. The small number of hydrogens involved can explain the difficulty in detecting this change by other methods.     

Tritium exchange of spectrin versus temperature

Tritium-exchange experiments were performed to assess the dynamics of the spectrin molecule. From the kinetics presented, spectrin appears as a fast-exchanging protein compared to globular proteins. This is easily explained by its known elongated structure. Spectrin tritium exchange is also shown to be highly temperature sensitive, even in buffer conditions that greatly reduce the temperature dependence of hydroxyl catalyst concentration. At high ionic strength, the exchange rates of specific hydrogens deduced from a power-law simulation are shown to exhibit an Arrhenius behavior, with enthalpies ranging from 70 to 140 kjmol^?1. These results are discussed in relation to the known high α-helix content of spectrin. Finally, a molecular model of α-helix opening is proposed that provides satisfactory agreement with the totality of the spectrin behavior in tritium-exchange experiments.     

The peptidyl prolyl cis/trans isomerase Pin1 downregulates the Inhibitor of Apoptosis Protein Survivin

The peptidyl prolyl cis-trans isomerase Pin1 and the Inhibitor of Apoptosis Protein (IAP) Survivin are two major proteins involved in cancer. They both modulate apoptosis, mitosis, centrosome duplication and neuronal development but until now no functional relationship has been reported between these two proteins. We tested Pin1-induced regulation of Survivin in neuroblastoma cells. Pin1 overexpression in SY5Y neuroblastoma cells decreased Survivin levels. Immunocytochemical studies indicated that they partially co-localized in interphase and mitotic cells. Co-immunoprecipitation further demonstrates the existence of a Pin1/Survivin complex. Pin1-induced effect on Survivin was confirmed in COS cells. RT-PCR and mutagenesis experiments suggested that this Pin1-induced decrease of Survivin occurred at the protein level. Survivin downregulation depended on the binding ability of Pin1 but was not related to the single Thr-Pro site, suggesting an indirect relationship into a protein complex. Finally, this functional regulation of Survivin by Pin1 is reciprocal since Pin1 silencing led to an increase in Survivin levels. The characterization of this functional relationship between Pin1 and Survivin might help to better understand mitosis control and cancer mechanisms.     

Protons and Mg2+ cations as probes in investigating the role of GTP in initiation complex formation.

fMet-tRNAfMet binding to both 30-S subunits and to 70-S particles is dependent on both pH AND Mg2+ concentration: for fMet-tRNAfMet binding to 70-S particles, variations of pH and Mg2+ concentration are tightly interdependent. This behavior can be interpreted by the polyelectrolyte theory as a direct consequence of the fact that the binding occurs in a polyanionic micro-environment. The pH-dependent binding to 70-S particles clearly shows the involvement of two prototropic groups which appear to be those carrying out GTP hydrolysis, therefore directly linked to initiation complex formation; in the presence of a non-hydrolyzable analogue to GTP, guanosine 5'-[beta, gamma-imido]triphosphate, the binding of fMet-tRNAfMet shows much less interdependence between variation of pH and Mg2+ concentration.     

Interaction of anilinonaphtyl labeled spectrin with fatty acids and phospholipids: a fluorescence study

Anilinonaphtyl labeled spectrin exhibits a fluorescence emission spectrum characteristic of a highly hydrophobic environment. Quenching of the fluorescence intensity by nitroxide analogs of fatty acids of affinity 10(4) M-1 reveals that the sites of interaction of fatty acids lie very close to the anilinonaphtyl groups. Similar experiments performed with a nitroxide analog of phosphatidylserine yield a 30% quenching of fluorescence while the same phosphatidylcholine analog has essentially no effect. The changes in the fluorescence emission spectrum exhibited in the presence of sonicated phosphatidylserine vesicles further outline the specificity of interaction towards phosphatidylserine, with one spectrin binding site per about 750 exposed phospholipids. Moreover, they suggest a penetration of the anilinonaphtyl group into the lipid bilayer.