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

Biophys J. 2004 Aug;87(2):1205-14. doi: 10.1529/biophysj.103.038844.

Abstract

We report the backbone dynamics of monomeric phospholamban in dodecylphosphocholine micelles using (1)H/(15)N 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 approximately 80 degrees (Zamoon et al., 2003; Mascioni et al., 2002). T(1), T(2), and (1)H/(15)N 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 micros-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 micros-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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Calcium-Binding Proteins / chemistry*
  • Colloids / chemistry*
  • Computer Simulation
  • Membrane Proteins / chemistry
  • Micelles
  • Models, Molecular*
  • Nitrogen Isotopes
  • Nuclear Magnetic Resonance, Biomolecular / methods*
  • Phosphorylcholine / analogs & derivatives*
  • Phosphorylcholine / chemistry*
  • Protein Conformation
  • Protein Structure, Tertiary
  • Protons

Substances

  • Calcium-Binding Proteins
  • Colloids
  • Membrane Proteins
  • Micelles
  • Nitrogen Isotopes
  • Protons
  • phospholamban
  • Phosphorylcholine
  • dodecylphosphocholine