Estabilidad conformacional e interaccion con lipidos en dos sistemas modelo samp73, una proteina soluble monomerica, y kcsa, una proteina de membrana tetramerica

Resumen

The human p73 protein is a transcription factor involved in apoptosis control and nervous system development, being homologous to the important tumour suppressor protein p53. p73 contains a SAM domain (SAMp73) in its C-terminus, whose stability and function have not been characterized. SAMp73 is composed of 65 amino acids, has helical structure, is water-soluble and monomeric. Fluorescence studies determined that the conformation of SAMp73 does not change importantly with the pH. However, transitions at acid and basic pH, likely corresponding to local changes, were monitored. Stability studies allowed to determine that SAMp73 folds through a two-state mechanism, with a maximum stability in the pH range from 6 to 10 (deltaG 5.7 kcal/mol). While this value is only moderate, the domain shows a high thermo-resistance (Tm 93.5 °C), due to a small unfolding heat capacity change. On the other hand, in order to determine whether the function of SAMp73 could be related to the binding in vivo to lipid membranes, we performed biophysical experiments and theoretical predictions on the interaction of this domain with lipids. We determined experimentally that SAMp73 has the ability to bind to the surface of lipid membranes, showing a higher affinity for zwitterionic lipids (phosphatidyl choline) than for ainionic lipids (phosphatidic acid). The lipid-binding causes a moderate increase in the alpha helical content of the protein and the solvent exposition of the only tryptophan. The use of the theoretical scales of hydrophobicity and interfaciality (wimley, WC and White SH, 1996, Experimentally determined hydrophobicity scale for proteins at membrane interfaces. Nat. Struct. Biol. 3: 842-848) predicted that the protin-lipid interaction occurs through three different regions distant in the amino acids sequence, but that seem to form a continuous surface in SAMp73.%&/The potassium channel KcsA is an integral membrane protein which functions as a potassium channel. The protein, from the soil bacterium Streptomyces lividans, is structured as a tetramer, being each monomer, of 160 amino acids, composed by two transmembrane helices, and cytoplasmatic N- and C-terminal domains. The protein is able to self-associate, forming clusters which importantly modulate its function. For KcsA reconstituted in asolectin vesicles, it seems that most of the channels are forming part of these clusters: however, for the protein solubilized in detergent (DDM), only 5-10% of the channels assemble in clusters. The KcsA tetramer is highly stable, being resistant to high temperatures and to high concentrations of the commonly employed denaturing agents SDS, urea and guanidinium chloride. However, KcsA is unfolded by the alcohol 2,2,2-trifluoroethanol through three successive sigmoidal transitions at neutral pH. In the first transition, the oligomeric structures formed by several channels are dissociated without important changes neither in the secondary structure nor in the tertiary structure of KcsA. Higher TFE concentrations led to the monomerization of the protein and an important loss of secondary structure (from 70% alpha helix to 25% alpha helix). During the third transition, ocurring at high concentrations of TFE, the monomers adopted a non-native, highly helical structure and aggregate. The last transition is totally irreversible, while the second transition is partially reversible when the tprotein is solubilized in the detergent dodecylmaltoside. The first of the three described transitions seems also to be highly reversible. The presence of the lipids DOPE and DOPG led to an important increase in the reversibility of the second (unfolding and monomerization) transition, as determined by SDS-PAGE and fluorescence experiments. This allowed to characterize that the monomerization of KcsA occurs without the presence of intermediates significantly populated in the equilibrium, and with a stability of 30 +- 3 kcal/mol. These results highlight ther role of selected lipids as effectors in the correct folding of KcsA, while their influence in the formation of clusters seems to be more structural, as the presence of a stable bilayer is required.