Lipid-protein and protein-protein interactions in the mechanisms of photosynthetic reaction centre and the Na+,K+-ATPase

Abstract

Lipid-protein and protein-protein interactions are likely to play important roles in the function and regulation of charge-transporting membrane proteins. This thesis focuses on two different membrane proteins, the photosynthetic reaction centre (RC) from purple bacteria and the Na+,K+-ATPase. The influence of the lipid surroundings and cholesterol derivatives on the kinetics of electron transfer of the RC were investigated by reconstituting the protein in phosphatidylcholine vesicles containing cholesterol and derivatives known to modulate the membrane dipole potential. The experiments performed on the Na+,K+-ATPase were designed to contribute to a better understanding of the role that oligomeric protein-protein interactions have in the enzyme’s mechanism. Our results show that the cholesterol derivatives significantly modify the electron transfer kinetics within the RCs and their multiphasic behavior. These effects seem to be associated with the extent of the dipole potential change experienced by the RC within the phospholipid membrane. Indeed, the largest effects on the rates are observed when 6-ketocholestanol and cholesterol are present, consistent by with their previously demonstrated significant increase of the dipole potential. We interpret this data as indicating an increased free energy barrier for protons to enter the protein. The consequences of the increased dipole potential seem to be experienced across the entire protein, since the rates of the P+QA- charge recombination in the presence of AQ- acting as QA are also modified by the same effectors. Also interesting is the effect of the dipole potential on the two conformational states of the RCs (previously reported) as revealed by the biphasic decays of the electron transfer kinetics. In particular, we report for the first time a biphasicity of the P+QA- charge recombination in the WT RCs. This non exponential behaviour, absent in the phospholipid membrane or isolated RCs, is induced by the presence of the cholesterol derivatives, suggesting that the equilibration time between the two RC conformations is slowed down significantly by these molecules. According to this work, the dipole potential seems to be an important parameter that has to be taken into account for a fine understanding of the charge transfer function of the RCs. Reported literature values of the dissociation constant, Kd, of ATP with the E1 conformation of the Na+,K+-ATPase based on equilibrium titrations and kinetic methods disagree. Using isothermal titration calorimetry (ITC) and simulations of the expected equilibrium behaviour for different binding models, this thesis presents an explanation for this apparent discrepancy based on protein-protein interactions. Because of the importance of Mg2+ in ATP hydrolysis, kinetic studies of Mg2+ binding to the protein were also carried out. These studies showed that ATP alone is responsible for Mg2+ complexation, with no significant contribution from the enzyme environment