Binding of uniformly 13 C labelled ATP to Na,KATPase was studied by 13 C cross-polarization magic-angle spinning (CP-MAS) NMR. In the presence of 30 mM Na + , and with sample-and time-averaging, NMR spectra obtained at 4°C exhibited several resonances for the bound nucleotide. Chemical shifts suggested that site-specific changes in the micro-environment or conformation of the nucleotide occurred in the high affinity binding site. These experiments permit further studies of nucleotide dynamics, structure and binding under physiologically relevant conditions.

The structure of a synthetic peptide corresponding to the fifth membrane-spanning segment (M5) in Na + ,K + -ATPase in sodium dodecyl sulfate (SDS) micelles was determined using liquid-state nuclear magnetic resonance (NMR) spectroscopy. The spectra reveal that this peptide is substantially less a-helical than the corresponding M5 peptide of Ca 2+ -ATPase. A well-defined a-helix is shown in the C-terminal half of the peptide. Apart from a short helical stretch at the N-terminus, the N-terminal half contains a non-helical region with two proline residues and sequence similarity to a non-structured transmembrane element of the Ca 2+ -ATPase. Furthermore, this region spans the residues implicated in Na + and K + transport, where they are likely to offer the flexibility needed to coordinate Na + as well as K + during active transport.

Cation binding to Na,K-ATPase is characterized in native membranes at room temperature by solid-state NMR spectroscopy using the K(+) congener (205)Tl. It has been demonstrated that the signals from occluded Tl(+) and nonspecifically bound Tl(+) can be detected and distinguished by NMR. Effects of dipole-dipole coupling between (1)H and (205)Tl in the occlusion sites show that the ions are rigidly bound, rather than just occluded. Furthermore, a low chemical shift suggests occlusion site geometries with a relatively small contribution from carboxylate and hydroxyl groups. Nonspecific binding of Tl(+) is characterized by rapid chemical exchange, in agreement with the observed low binding affinity.

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