Probing excited states and activation energy for the integral membrane protein phospholamban by NMR CPMG relaxation dispersion experiments.
Phospholamban (PLN) is a dynamic single-pass membrane protein that inhibits the flow of Ca(2+) ions into the sarcoplasmic reticulum (SR) of heart muscle by directly binding to and inhibiting the SR Ca(2+)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 mus 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 (15)N backbone and (13)C(delta1) 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 mus-ms dynamics (k(ex)=6100+/-800 s(-1) at 17 degrees C). We measured k(ex) at additional temperatures, which allowed for a calculation of activation energy equal to approximately 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.
Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, Minneapolis, MN 55445, USA.
Nathaniel J
NJ
Traaseth
Gianluigi
G
Veglia
D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D003902:Detergents / Q000737:chemistry; D006801:Humans; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry*; D008823:Micelles; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D017434:Protein Structure, Tertiary / Q000502:physiology; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism; D000099211:Phospholamban
D002135:Calcium-Binding Proteins; D003902:Detergents; D008051:Lipid Bilayers; D008565:Membrane Proteins; D008823:Micelles; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban