TNP-AMP binding to the sarcoplasmic reticulum Ca(2+)-ATPase studied by infrared spectroscopy.
Infrared spectroscopy was used to monitor the conformational change of 2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-monophosphate (TNP-AMP) binding to the sarcoplasmic reticulum Ca(2+)-ATPase. TNP-AMP binding was observed in a competition experiment: TNP-AMP is initially bound to the ATPase but is then replaced by beta,gamma-iminoadenosine 5'-triphosphate (AMPPNP) after AMPPNP release from P(3)-1-(2-nitrophenyl)ethyl AMPPNP (caged AMPPNP). The resulting infrared difference spectra are compared to those of AMPPNP binding to the free ATPase, to obtain a difference spectrum that reflects solely TNP-AMP binding to the Ca(2+)-ATPase. TNP-AMP used as an ATP analog in the crystal structure of the sarcoplasmic reticulum Ca(2+)-ATPase was found to induce a conformational change upon binding to the ATPase. It binds with a binding mode that is different from that of AMPPNP, ATP, and other tri- and diphosphate nucleotides: TNP-AMP binding causes partially opposite and smaller conformational changes compared to ATP or AMPPNP. The conformation of the TNP-AMP ATPase complex is more similar to that of the E1Ca(2) state than to that of the E1ATPCa(2) state. Regarding the use of infrared spectroscopy as a technique for ligand binding studies, our results show that infrared spectroscopy is able to distinguish different binding modes.
Institut für Biophysik, Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany.
Man
M
Liu
Andreas
A
Barth
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D001667:Binding, Competitive; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism; D046911:Macromolecular Substances; D008968:Molecular Conformation; D011485:Protein Binding; D011487:Protein Conformation; D012519:Sarcoplasmic Reticulum / Q000737:chemistry* / Q000201:enzymology* / Q000378:metabolism; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*
D046911:Macromolecular Substances; D000249:Adenosine Monophosphate; C036744:2',3'-O (2,4,6-trinitrocyclohexadienylidine)adenosine 5'-monophosphate; D000252:Calcium-Transporting ATPases