Structural dynamics and topology of phospholamban in oriented lipid bilayers using multidimensional solid-state NMR.
Phospholamban (PLN), a single-pass membrane protein, regulates heart muscle contraction and relaxation by reversible inhibition of the sarco(endo)plasmic reticulum Ca-ATPase (SERCA). Studies in detergent micelles and oriented lipid bilayers have shown that in its monomeric form PLN adopts a dynamic L shape (bent or T state) that is in conformational equilibrium with a more dynamic R state. In this paper, we use solid-state NMR on both uniformly and selectively labeled PLN to refine our initial studies, describing the topology and dynamics of PLN in oriented lipid bilayers. Two-dimensional PISEMA (polarization inversion spin exchange at the magic angle) experiments carried out in DOPC/DOPE mixed lipid bilayers reveal a tilt angle of the transmembrane domain with respect to the static magnetic field, of 21 +/- 2 degrees and, at the same time, map the rotation angle of the transmembrane domain with respect to the bilayer. PISEMA spectra obtained with selectively labeled samples show that the cytoplasmic domain of PLN is helical and makes an angle of 93 +/- 6 degrees with respect to the bilayer normal. In addition, using samples tilted by 90 degrees , we find that the transmembrane domain of PLN undergoes fast long-axial rotational diffusion about the bilayer normal with the cytoplasmic domain undergoing this motion and other complex dynamics, scaling the values of chemical shift anisotropy. While this dynamic was anticipated by previous solution NMR relaxation studies in micelles, these measurements in the anisotropic lipid environment reveal new dynamic and conformational features encoded in the free protein that might be crucial for SERCA recognition and subsequent inhibition.
Traaseth
N J
NJ
Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, USA.
Buffy
J J
JJ
Zamoon
J
J
Veglia
G
G
D002135:Calcium-Binding Proteins / Q000737:chemistry*; D008051:Lipid Bilayers; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D000099211:Phospholamban
D002135:Calcium-Binding Proteins; D008051:Lipid Bilayers; D000099211:Phospholamban