ABSTRACT

This PhD study is carried out at the Department of Biophysics at Aarhus University and deals with the Na,K-ATPase, which is a membrane-bound enzyme transporting 3 Na + out of and 2 K + into the cell against their electrochemical gradients at the expense of one ATP. This enzyme belongs to the P-type ATPase family, of which only structures of the Ca-ATPase in certain conformations are known. Structural knowledge is important for understanding the enzyme mechanism. Solid-state NMR does not require crystallization or fast molecular motion of the enzyme as is the case for X-ray diffraction or liquid-state NMR, respectively, and may therefore be a suitable method for elucidation of ligand-protein interactions.

The K + -occluding state, E 2 K 2 , is spontaneously formed in the presence of K + or a congener, e.g. Tl + , Rb + , or Cs + . Fluorescence and equilibrium binding experiments have revealed a specific Tl + binding eight times stronger than the corresponding K + binding. Together with a high NMR sensitivity and a vast chemical shift area, the 205 Tl nucleus seems ideal for NMR investigations, which reflects interactions of the cation and the local chemical surroundings. Solid-state 205 Tl NMR experiments at a low concentration of Tl + , in which the specific binding is dominating, show a narrow signal with no chemical shift anisotropy line shape, indicating that the cations in the binding sites of the Na,K-ATPase are not tightly bound. A clear effect of 1 H decoupling suggests that the occluded cations are not freely mobile either. Thus, occluded cations experience a limited degree of mobility. This is in contrast to the situation with the Ca-ATPase, where occluded cations are tightly coordinated. Both NMR spectra and binding experiments indicate two different kinds of non-specific binding sites.

As ATP is the energy source of the active transport, knowledge of the nucleotide binding site structure is important. Preliminary 13 C cross-polarization magic angle spinning NMR experiments are performed with the purpose of describing binding of nucleotide to the enzyme and determine the binding and dissociation rate constants.

The fifth transmembrane span, M5, in the Na,K-ATPase is important in coordination of the cation occlusion sites, and the structure of this peptide solubilized in SDS micelles has been investigated with liquid-state NMR. Chemical shift and NOE connectivity analyses, which are supported by CD-spectra and structure prediction, indicate a predominantly β -sheet structure, which is in contrast to the Ca-ATPase M5 consisting of two α -helices and a kink. This difference may be related to the fact that the Na,K-ATPase binds 3 Na + where the Ca-ATPase only binds 2 Ca 2+ ions.