1H/15N heteronuclear NMR spectroscopy shows four dynamic domains for phospholamban reconstituted in dodecylphosphocholine micelles

Emily E. Metcalfe, Jamillah Zamoon, David D. Thomas, Gianluigi Veglia

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We report the backbone dynamics of monomeric phospholamban in dodecylphosphocholine micelles using 1H/15N heteronuclear NMR spectroscopy. Phospholamban is a 52-amino acid membrane protein that regulates Ca-ATPase in cardiac muscle. Phospholamban comprises three structural domains: a transmembrane domain from residues 22 to 52, a connecting loop from 17 to 21, and a cytoplasmic domain from 1 to 16 that is organized in an "L"-shaped structure where the transmembrane and the cytoplasmic domain form an angle of ∼80° (Zamoon et al., 2003; Mascioni et al., 2002). T1, T2, and 1H/15N nuclear Overhauser effect values measured for the amide backbone resonances were interpreted using the model-free approach of Lipari and Szabo. The results point to the existence of four dynamic domains, revealing the overall plasticity of the cytoplasmic helix, the flexible loop, and part of the transmembrane domain (residues 22-30). In addition, using Carr-Purcell-Meiboom-Gill-based experiments, we have characterized phospholamban dynamics in the μs-ms timescale. We found that the majority of the residues in the cytoplasmic domain, the flexible loop, and the first ten residues of the transmembrane domain undergo dynamics in the μs-ms range, whereas minimal dynamics were detected for the transmembrane domain. Hydrogen/deuterium exchange factors measured at different temperatures support the existence of slow motion in both the loop and the cytoplasmic helix. We propose that these dynamic properties are critical factors in the biomolecular recognition of phospholamban by Ca-ATPase and other interacting proteins such as protein kinase A and protein phosphatase 1.

Original languageEnglish (US)
Pages (from-to)1205-1214
Number of pages10
JournalBiophysical journal
Issue number2
StatePublished - Aug 2004

Bibliographical note

Funding Information:
NMR instrumentation was provided with funds from the National Science Foundation (BIR-961477) and the University of Minnesota Medical School. This work was supported in part by grants to G.V. (National Institutes of Health grant GM64742; American Heart Association grant 0160465Z), and D.D.T. (National Institutes of Health grant GM27906, University of Minnesota Academic Health Center).


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