orals 5th Asia-Pacific NMR Symposium 2013

Solid-state NMR Spectra of Pseudo-GPI-anchored Proteins in Membranes under Magic Angle Spinning (#16)

Kaoru Nomura 1 , Erisa Harada 1 , Kenji Sugase 1 , Keiko shimamoto 1
  1. Bioorganic Research Institute, Suntory Foundation for life sciences, Shimamoto-Cho, Osaka, Japan

We developed a methodology to observe a high resolution solid-state NMR spectra for a Glycosylphosphatidylinisotol (GPI)-anchored protein embedded in membranes under magic angle spinning (MAS) for the first time. GPI-anchored proteins are cell surface proteins in which the C-terminus is covalently linked to a GPI anchor consisting of a phosphoethanolamine linker, a glycan core, and a phospholipid tail. Although GPI-anchored proteins play diverse functions similar to other membrane proteins, atomic level analyses of their 3D structures, dynamics, and membrane interactions have not been achieved yet. This is because supplying sufficient amount of stable isotope labels in overexpression is difficult for GPI-anchored proteins which require complex posttranslational modification. In this study, we designed a new type of pseudo-GPI-anchored protein in which the protein part was expressed in E. coli and attached to a chemically synthesized GPI anchor mimic. Using two different types of membranes, liposomes and bicelles, we demonstrated two types of insertion procedures for GPI-anchored protein into membranes.
  Solid-state NMR were measured for both samples under 5 kHz MAS. 1D and 2D refocused INEPT and 1D cross polarization (CP) spectra were successfully obtained. The bicelle sample showed sufficient quality of 1D and 2D INEPT spectra due to their high membrane fluidity. It suggests that bicelle samples are applicable for NMR signal assignments, 3D structure determination, and dynamics studies of larger GPI-anchored protein samples. In contrast, in liposome samples, both the protein and lipid peaks were observed in CPMAS spectrum at low temperature, suggesting the possibility to analyze inter- and intra-molecular interactions in dipolar coupling experiments. Thus, this study demonstrated the feasibility of this methodology to examine the roles of GPI-anchor in regulating diverse biological phenomena such as embryonic development, immune responses, and diseases related to GPI-anchored proteins.