Understanding the van der Waals forces that mediate quaternary interactions in membrane proteins

Integral membrane proteins (MPs) play major roles in facilitating molecular trafficking and cellular communication, yet little is known about how their activity is mediated. One common regulatory mechanism observed for many MPs is oligomerization, which can increase the requirements for protein activity. Thus, understanding the forces that drive MP interactions can allow for a greater understanding of how MPs mediate function. Previous studies designed to determine the contributions of hydrogen bonds and van der Waals forces to membrane protein-protein interactions suggested that van der Waals interactions contributed a dominating force to association. In this thesis, we further investigated the influence of van der Waals forces on quaternary interactions by utilizing two diverse systems: the α-helical transmembrane domain of the erythropoietin receptor (EpoR) from mouse and human, and the β-barrel Outer Membrane Phospholipase A (OMPLA) from E.coli. To elucidate how changes in van der Waals interactions influence oligomerization in these systems, we used sedimentation equilibrium analytical ultracentrifugation to measure the change in free energy of association incurred for detergent-solubilized MPs upon acquiring sequence mutations that perturb packing interactions. With EpoR, we compared the change in free energy of association of the human transmembrane domain to that of the mouse. We found that a leucine zipper motif present in both sequences drives self-association of the transmembrane domains, although to a lesser extent in the human sequence (by ∼0.9 kcal mol-1) where the motif is shortened. In both cases, however, the association was not as energetically favorable as implied by previous research. Our data suggest that the propensity for self-association previously observed is likely a combination of interactions between the extracellular, intracellular and transmembrane domains of the native receptor. In the β-barrel OMPLA, we investigated the influence of packing interactions on dimerization. Using sequence variants designed to perturb packing interactions at the dimer interface, we determined that there is no correlation between loss of occluded surface area and loss of association free energy. This is in stark contrast to the prevailing view for α-helix interactions. Taken together, these data imply that MP interactions are more complex than the current theoretical models allow