title: Mapping interactions between the Ca2+-ATPase and its substrate ATP with infrared spectroscopy.
abstract: Infrared spectroscopy has been used to map substrate-protein interactions: the conformational changes of the sarcoplasmic reticulum Ca(2+)-ATPase upon nucleotide binding and ATPase phosphorylation were monitored using the substrate ATP and ATP analogues (2'-deoxy-ATP, 3'-deoxy-ATP, and inosine 5'-triphosphate), which were modified at specific functional groups of the substrate. Modifications to the 2'-OH, the 3'-OH, and the amino group of adenine reduce the extent of binding-induced conformational change of the ATPase, with particularly strong effects observed for the latter two. This demonstrates the structural sensitivity of the nucleotide-ATPase complex to individual interactions between nucleotide and ATPase. All groups studied are important for binding and interactions of a given ligand group with the ATPase depend on interactions of other ligand groups. Phosphorylation of the ATPase was observed for ITP and 2'-deoxy-ATP, but not for 3'-deoxy-ATP. There is no direct link between the extent of conformational change upon nucleotide binding and the rate of phosphorylation showing that the full extent of the ATP-induced conformational change is not mandatory for phosphorylation. As observed for the nucleotide-ATPase complex, the conformation of the first phosphorylated ATPase intermediate E1PCa(2) also depends on the nucleotide, indicating that ATPase states have a less uniform conformation than previously anticipated.
authors.affiliation: Institut für Biophysik, Johann Wolfgang Goethe-Universität, Theodor-Stern-Kai 7, Haus 74, D-60590 Frankfurt am Main, Germany.
authors.forename: Man
authors.initials: M
authors.lastname: Liu
authors.forename: Andreas
authors.initials: A
authors.lastname: Barth
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases