The Use of Metal Fluoride Compounds as Phosphate Analogs for Understanding the Structural Mechanism in P-type ATPases.

The membrane-bound protein family, P-type ATPases, couples ATP hydrolysis with substrate transport across the membrane and forms an obligatory auto-phosphorylated intermediate in the transport cycle. The metal fluoride compounds, BeF x , AlF x , and MgF x , as phosphate analogs stabilize different enzyme structural states in the phosphoryl transfer/hydrolysis reactions, thereby fixing otherwise short-lived intermediate and transient structural states and enabling their biochemical and atomic-level crystallographic studies. The compounds thus make an essential contribution for understanding of the ATP-driven transport mechanism. Here, with a representative member of P-type ATPase, sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA), we describe the method for their binding and for structural and functional characterization of the bound states, and their assignments to states occurring in the transport cycle.