The overall objective is to understand the conformational mobility of various segments of the a-subunit during the reaction cycle. The a-subunit of the Na pump has a large cytoplasmic loop which contains all amino acids that are implicated in ATP binding or phosphorylation. This proposal will obtain detailed structural data on this domain, which is now available in isolation from a bacteria over-expression system. Strong evidence exists to support the present line of experimentation. The investigator has found that, upon incubation of the intact protein in the absence of K ions, the M5-M6 peptide was lost from the membrane to the aqueous phase. The post-tryptic residue was still able to occlude K ions.
The specific aim here is to obtain large quantities of this ATP-binding domain for structural and mutagenesis studies. Functional expression of the Na/K ATPase in a heterologous expression system is still in its infancy. This proposal will exploit the potential of mutagenesis and heterologous expression to obtain structural and mechanistic information about the Na pump. The recent achievements of the investigator's laboratory in obtaining good functional levels of Na/K ATPase in Hi5 cells following low titer infection with baculovirus heralds a significant and exciting change in protocols during the current funding period.
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