Determination of structural topology of a membrane protein in lipid bilayers using polarization optimized experiments (POE) for static and MAS solid state NMR spectroscopy

作者：Mote Kaustubh R; Gopinath T; Veglia Gianluigi^*

来源：Journal of Biomolecular NMR, 2013, 57(2): 91-102.

DOI：10.1007/s10858-013-9766-2

• Oriented solid-state NMR (OSS-NMR)
• Magic angle spinning solid state NMR
• Membrane proteins
• DUMAS-ssNMR
• MEIOSIS
• Sarcolipin
• Magnetically aligned bicelles
• Hybrid method for membrane protein structure determination

The low sensitivity inherent to both the static and magic angle spinning techniques of solid-state NMR (ssNMR) spectroscopy has thus far limited the routine application of multidimensional experiments to determine the structure of membrane proteins in lipid bilayers. Here, we demonstrate the advantage of using a recently developed class of experiments, polarization optimized experiments, for both static and MAS spectroscopy to achieve higher sensitivity and substantial time-savings for 2D and 3D experiments. We used sarcolipin, a single pass membrane protein, reconstituted in oriented bicelles (for oriented ssNMR) and multilamellar vesicles (for MAS ssNMR) as a benchmark. The restraints derived by these experiments are then combined into a hybrid energy function to allow simultaneous determination of structure and topology. The resulting structural ensemble converged to a helical conformation with a backbone RMSD similar to 0.44 , a tilt angle of 24A degrees A A +/- A 1A degrees, and an azimuthal angle of 55A degrees A A +/- A 6A degrees. This work represents a crucial first step toward obtaining high-resolution structures of large membrane proteins using combined multidimensional oriented solid-state NMR and magic angle spinning solid-state NMR.

• 出版日期2013-10