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

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

ATP Binding Equilibria of the Na+,K+-ATPase

Reported values of the dissociation constant, Kd, of ATP with the E1 conformation of the Na+,K+-ATPase fall in two distinct ranges depending on how it is measured. Equilibrium binding studies yield values of 0.1-0.6 μM, whereas presteady-state kinetic studies yield values of 3-14 μM. It is unacceptable that Kd varies with the experimental method of its determination. Using simulations of the expected equilibrium behavior for different binding models based on thermodynamic data obtained from isothermal titration calorimetry we show that this apparent discrepancy can be explained in part by the presence in presteady-state kinetic studies of excess Mg2+ ions, which compete with the enzyme for the available ATP. Another important contributing factor is an inaccurate assumption in the majority of presteady-state kinetic studies of a rapid relaxation of the ATP binding reaction on the time scale of the subsequent phosphorylation. However, these two factors alone are insufficient to explain the previously observed presteady-state kinetic behavior. In addition one must assume that there are two E1-ATP binding equilibria. Because crystal structures of P-type ATPases indicate only a single bound ATP per R-subunit, the only explanation consistent with both crystal structural and kinetic data is that the enzyme exists as an (αβ)2 diprotomer, with protein-protein interactions between adjacent R-subunits producing two ATP affinities. We propose that in equilibrium measurements the measured Kd is due to binding of ATP to one R-subunit, whereas in presteadystate kinetic studies, the measured apparent Kd is due to the binding of ATP to both R-subunits within the diprotomer

Mechanism of Mg2+ Binding in the Na+,K+-ATPase

The Mg2+ dependence of the kinetics of the phosphorylation and conformational changes of Na+,K+-ATPase was investigated via the stopped-flow technique using the fluorescent label RH421. The enzyme was preequilibrated in buffer containing 130 mM NaCl to stabilize the E1(Na+)3 state. On mixing with ATP, a fluorescence increase was observed. Two exponential functions were necessary to fit the data. Both phases displayed an increase in their observed rate constants with increasing Mg2+ to saturating values of 195 (± 6) s−1 and 54 (± 8) s−1 for the fast and slow phases, respectively. The fast phase was attributed to enzyme conversion into the E2MgP state. The slow phase was attributed to relaxation of the dephosphorylation/rephosphorylation (by ATP) equilibrium and the buildup of some enzyme in the E2Mg state. Taking into account competition from free ATP, the dissociation constant (Kd) of Mg2+ interaction with the E1ATP(Na+)3 state was estimated as 0.069 (± 0.010) mM. This is virtually identical to the estimated value of the Kd of Mg2+-ATP interaction in solution. Within the enzyme-ATP-Mg2+ complex, the actual Kd for Mg2+ binding can be attributed primarily to complexation by ATP itself, with no apparent contribution from coordination by residues of the enzyme environment in the E1 conformation