Characterization of membrane protein active transport under native-like conditions

Sammanfattning: P-type ATPase proteins are a family of membrane proteins that maintain concentration gradients of e.g. ions by ATP-driven transport across the membrane. While these transporters share many features in their molecular architecture, structural differences are required to convey ion specificity. In addition, the transport dynamics accomplished by conformational changes may also differ in-between ATPase subtypes. Therefore, resolving P-type ATPase temporal and spatial structural dynamics is crucial to understand how these proteins function.  To pave way for time-resolved X-ray characterization of conformational changes during P-type ATPase transport in solution, it was necessary to identify optimal conditions for triggering the protein reaction. Therefore, ATP activation of a recombinant Zn2+-transporting ATPase was studied using a biochemical activity assay and infrared spectroscopic techniques. Specifically, time-dependent Fourier-Transform Infra-Red (FTIR) spectroscopy was used to study activation using photolysis of caged ATP. The highest protein activity was obtained at a protein concentration of 25 mg/mL at 310 K and pH 7, and this required the presence of 20% glycerol as a stabilizing agent. It was also observed that neither the presence of caged ATP nor higher lipid concentrations affected protein activity significantly.  The Ca2+-transporting sarcoplasmic reticulum ATPase (SERCA), found abundantly in skeletal muscle native membranes, was used to develop the time-resolved Wide-Angle X-ray Scattering (TR-WAXS) technique for irreversible caged ATP activation and subsequent structural refinement. Several SERCA intermediate states and protein-lipid interactions have been characterized by X-ray crystallography, rendering the SERCA protein an ideal proof-of-principle target system. In the native membrane, fast single-cycle dynamics were registered followed by steady state accumulation. The structural refinement procedure starting from existing intermediate crystal structures indicated that the accumulated state represented a phosphorylated state (E2-P) or possibly a Ca2+ bound E2 state (Ca2E2P), which has so far eluded X-ray crystallographic characterization. The results also showed that the corresponding ground state (Ca2E1) underwent significant rearrangements of the cytosolic domains, which implies that the Ca2E1 crystal structure might be one of several possible structures and might not represent the dominant structure in solution. Additionally, the TR-WAXS models indicated that the rocking motion of the soluble domains observed in a detergent/lipid mixture is also present in the native membrane.