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TNP-AMP Binding to the Sarcoplasmic Reticulum Ca²⁺-ATPase Studied by Infrared Spectroscopy

Man Liu ^* and Andreas Barth 

^* Institut für Biophysik, Johann Wolfgang Goethe-Universität, Frankfurt am Main, Germany; and Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm University, Stockholm, Sweden

Correspondence: Address reprint requests to Andreas Barth, Tel: 46-816-2452; Fax: 46-815-5597; E-mail: Andreas.Barth{at}dbb.su.se.

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 Ca²⁺-ATPase. TNP-AMP binding was observed in a competition experiment: TNP-AMP is initially bound to the ATPase but is then replaced by ß,-iminoadenosine 5'-triphosphate (AMPPNP) after AMPPNP release from P³-1-(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 Ca²⁺-ATPase. TNP-AMP used as an ATP analog in the crystal structure of the sarcoplasmic reticulum Ca²⁺-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 E1Ca₂ state than to that of the E1ATPCa₂ 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.

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