Solid-State NMR Spectra of Lipid-Anchored Proteins under Magic Angle Spinning

Solid-state NMR is a promising tool for elucidating membrane-related biological phenomena. We achieved the measurement of high-resolution solid-state NMR spectra for a lipid-anchored protein embedded in lipid bilayers under magic angle spinning (MAS). To date, solid-state NMR measurements of lipid-anchored proteins have not been accomplished due to the difficulty in supplying sufficient amount of stable isotope labeled samples in the overexpression of lipid-anchored proteins requiring complex posttranslational modification. We designed a pseudo lipid-anchored protein in which the protein component was expressed in E. coli and attached to a chemically synthesized lipid-anchor mimic. Using two types of membranes, liposomes and bicelles, we demonstrated different types of insertion procedures for lipid-anchored protein into membranes. In the liposome sample, we were able to observe the cross-polarization and the ¹³C–¹³C chemical shift correlation spectra under MAS, indicating that the liposome sample can be used to analyze molecular interactions using dipolar-based NMR experiments. In contrast, the bicelle sample showed sufficient quality of spectra through scalar-based experiments. The relaxation times and protein–membrane interaction were capable of being analyzed in the bicelle sample. These results demonstrated the applicability of two types of sample system to elucidate the roles of lipid-anchors in regulating diverse biological phenomena

Pharmacological antiarrhythmic treatment pre- and post-ablation.

<p>Pharmacological antiarrhythmic treatment pre- and post-ablation.</p

Identification of labeling of the left atrium (LA) in two-dimensional viewing.

<p>(a) LA top―mitral annulus diameter (dashed arrow). (b) LA longitudinal diameter (solid arrow). (c) LA anteroposterior diameter (solid arrow) and LA transverse diameter (dashed arrow).</p

Anatomical variation of PV in the PAF and non-PAF groups.

<p>Anatomical variation of PV in the PAF and non-PAF groups.</p

PV volume and LA volume in the non-recurrent and recurrent groups.

<p>PV volume and LA volume in the non-recurrent and recurrent groups.</p

Identification and labeling of the right superior pulmonary vein (RSPV) ostia in two-dimensional viewing.

<p>(a) Measurement of superoinferior diameter of RSPV ostium (solid arrow). (b) Measurement of anteroposterior diameter of RSPV ostium (solid arrow).</p

Clinical characteristics of patients with and without the recurrence of AF.

<p>Clinical characteristics of patients with and without the recurrence of AF.</p

Receiver-operating characteristics curve.

<p>Success after ablation of atrial fibrillation (AF) for pulmonary vein volume below 12.0 cm³ / BSA (m²) in the paroxysmal AF group.</p

Heart and great vessels, hardened in situ, posterior aspect.

<p>The superior Pulmonary veins (PVs) enter the left atrium (LA) from a superior, anterior, and lateral direction and the inferior PVs from the posterior direction. The right and left pulmonary arteries (PAs) lie just above and parallel to the right and left superior PVs. The ostium of the left inferior PV lies between the LA and descending aorta (Ao). There is neither artery nor bronchi (Br) adjacent to the right inferior PV. LSPV, left superior PV; LIPV. left inferior PV; RSPV, right superior PV; RIPV. right inferior PV.</p

Clinical characteristics of patients in the PAF and non-PAF groups.

<p>Clinical characteristics of patients in the PAF and non-PAF groups.</p