Probing excited states and activation energy for the integral membrane protein phospholamban by NMR CPMG relaxation dispersion experiments

Nathaniel J. Traaseth, Gianluigi Veglia

Research output: Contribution to journalArticle

Abstract

Phospholamban (PLN) is a dynamic single-pass membrane protein that inhibits the flow of Ca2+ ions into the sarcoplasmic reticulum (SR) of heart muscle by directly binding to and inhibiting the SR Ca2+ATPase (SERCA). The PLN monomer is the functionally active form that exists in equilibrium between ordered (T state) and disordered (R state) states. While the T state has been fully characterized using a hybrid solution/solid-state NMR approach, the R state structure has not been fully portrayed. It has, however, been detected by both NMR and EPR experiments in detergent micelles and lipid bilayers. In this work, we quantitatively probed the μs to ms dynamics of the PLN excited states by observing the T state in DPC micelles using CPMG relaxation dispersion NMR spectroscopy under functional conditions for SERCA. The 15N backbone and 13Cδ1 Ile-methyl dispersion curves were fit using a two-state equilibrium model, and indicate that residues within domain Ia (residues 1-16), the loop (17-22), and domain Ib (23-30) of PLN undergo μs-ms dynamics (kex = 6100 ±800 s- 1 at 17 °C). We measured kex at additional temperatures, which allowed for a calculation of activation energy equal to ∼ 5 kcal/mol. This energy barrier probably does not correspond to the detachment of the amphipathic domain Ia, but rather the energy needed to unwind domain Ib on the membrane surface, likely an important mechanism by which PLN converts between high and low affinity states for its binding partners.

Original languageEnglish (US)
Pages (from-to)77-81
Number of pages5
JournalBiochimica et Biophysica Acta - Biomembranes
Volume1798
Issue number2
DOIs
StatePublished - Feb 2010

Keywords

  • Ca-ATPase
  • Membrane Protein
  • NMR
  • Phospholamban
  • Protein Dynamics
  • Relaxation Dispersion
  • SERCA
  • Solution NMR

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Cell Biology

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