The distance between the ß-subunits of Na + /K + -ATPase isolated from pig dark red kidney medulla was determined by Forster energy transfer. First, oligosaccharides of the ß-subunit were shown to be labelled with three fluorophores: Lucifer yellow (LY), Lissamine rhodamine B sulfonyl hydrazine (LRSH) and Cascade blue (CB). Further, LY and LRSFI were used as the donor and the acceptor, respectively, for Forster energy transfer studies to determine the localization of the ß-subunit in the native enzyme which is known to be formed as a tetramer (aß)2. It was found that the ß-subunits in the functional enzyme complex in the membrane are not localized next to each other but are spatially separated. The distance between fluorophores covalently attached to the ß-subunits was found to be 5.1 nm. This conclusion was confirmed by measurements with another donor- acceptor pair CB-LY. The results also support the idea of a direct interaction of the ß-subunit with the extracellular part of the a-subunit. These interactions were modified in the presence of millimolar concentrations of magnesium ions. This indicates a crucial role of magnesium in extracellular interactions between the alpha and beta subunits.
Beside of the protein crystallography or NMR, another attractive option in protein structure analysis has recently appeared: computer modeling of the protein structure based on homology and similarity with proteins of already known structures. We have used the combination of computer modeling with spectroscopic techniques, such as steady-state or
time-resolved fluorescence spectroscopy, and with molecular biology techniques. This method could provide useful structural information in the cases where crystal or NMR structure is not available. Molecular modeling of the ATP site within the H4-H5-loop revealed eight amino acids residues, namely besides the previously reported amino acids Asp443, Lys480, Lys
501, Gly502 and Arg544, also Glu446, Phe475 and Gln 482, which form the complete ATP recognition site. Moreover, we have proved that a hydrogen bond between Arg423 and Glu472 supports the connection of two opposite halves of the ATP-binding pocket. Similarly, the conserved residue Pro489 is important for the proper interaction of the third and fourth
β-strands, which both contain residues that take part in the ATP-binding. Alternatively, molecular dynamics simulation combined with dynamic fluorescence spectroscopy revealed that 14-3-3 zeta C-terminal stretch is
directly involved in the interaction of 14-3-3 protein with the ligand. Phosphorylation at Thr232 induces a conformational change of the C-terminus, which is presumably responsible for observed inhibition of binding abilities. Phosphorylation at Thr232 induces more extended conformation of 14-3-3zeta C-terminal stretch and changes its interaction with the rest of the 14-3-3 molecule. This could explain negative regulatory effect of phosphorylation at Thr232 on 14-3-3 binding properties.