Determination of Membrane Protein Structure and Dynamics by Magic-Angle-Spinning Solid-State NMR Spectroscopy

Abstract

It is shown that molecular structure and dynamics of a uniformly labeled membrane protein can be studied under magic-angle-spinning conditions. For this purpose, dipolar recoupling experiments are combined with novel through-bond correlation schemes that probe mobile protein segments. These NMR schemes are demonstrated on a uniformly [13C,15N] variant of the 52-residue polypeptide phospholamban. When reconstituted in lipid bilayers, the NMR data are consistent with an alpha-helical trans-membrane segment and a cytoplasmic domain that exhibits a high degree of structural disorder.

Phospholamban and Its Phosphorylated Form Interact Differently with Lipid Bilayers: A31P,2H, and13C Solid-State NMR Spectroscopic Study

“…The two peaks are well within the range of an α-helical structure (see Table 1). The observed α-helical structure of the cytoplasmic domain of PLB agrees with the L-shaped (see Figure 1(A)) and the bellflower-like assembly high-resolution solution NMR structures (see Figure 1(C)) and disagrees with the non-α-helical disordered cytosolic domain model shown in Figure 1(B) (57,60,61).…”

“…Their data indicates that AFA-PLB has an Lshape structure and the cytosolic segment of AFA-PLB lies directly on the membrane surface (59,60). Another model for AFA-PLB proposed by the Baldus group, indicates a non-α-helical disordered cytosolic domain model for the monomer utilizing cross-polarization magic angle spinning (CPMAS) solid-state NMR 'spectroscopy (see Figure 1 (B)) (57). Finally, the Chou group reveals an unusual bellflower-like assembly for pentameric WT-PLB in micelles utilizing high-resolution solution NMR spectroscopy (61).…”

“…In both the L-shaped and the bellflower-like assembly solution NMR structures, the cytoplasmic domain is α-helical (59,61). The Baldus model suggests that the cytoplasmic domain of AFA-PLB is not α-helical (57). Additionally, while the L-shaped high-resolution solution NMR structure and the Middleton group studies suggest that the cytoplasmic domain interacts with the membrane surface (58,59,62), the Chou and the Baldus structures suggest that the cytoplasmic domain has minimal interactions with the membrane surface (phospholipid head groups) (57,61).…”

Structure and topology of monomeric phospholamban in lipid membranes determined by a hybrid solution and solid-state NMR approach

“…In fact, PLN does not possess a compact fold, but instead, has its 3D architecture dictated by interactions with the lipid bilayer. Several in vivo and in vitro studies have shown that PLN adopts a remarkably similar structure both in micelles and lipid bilayers (8,12,15,18,19,25,46,47), justifying the combination of restraints. In the event that restraints obtained in lipid bilayers and detergent micelles were incompatible, it is recommended that those from lipids be weighted more heavily, due to potential limitations of micelles.…”

Full-length Vpu and human CD4(372–433) in phospholipid bilayers as seen by magic angle spinning NMR

“…Dipolar coupling-based CP (Hartmann and Hahn, 1962;Pines et al, 1973) is effective if the vector connecting the interacting nuclei is restricted in mobility and correlation times of residual motions are above the millisecond range. In contrast, 1 H-13 C polarization transfer by insensitive nuclei enhanced by polarization transfer (INEPT) (Morris and Freeman, 1979;Burum and Ernst, 1980) is effective only if dipolar couplings are averaged out by large amplitude motions on time scales of < 10 -5 s (Andronesi et al, 2005;Yang et al, 2011). Slower motions in the range between milliseconds and microseconds on the other hand can affect T 1ρ relaxation and thus decrease the CP efficiency even if the amplitude of the motion is not large enough for isotropic averaging of the dipolar coupling (Fares et al, 2005;Sharpe et al, 2006).…”