Residual Dipolar Coupling Measurements of Transmembrane Proteins Using Aligned Low-q Bicelles and High-Resolution Magic Angle Spinning NMR Spectroscopy

摘要

Bicelles are a major medium form to produce weak alignment of soluble proteins for residual dipolar coupling (RDC) measurements. The obstacle to use the same type of bicelles for transmembrane proteins with solution-state NMR spectroscopy is the loss of signals due to the adhesion or penetration of the proteins into large bicelles, resulting in slow protein tumbling. In this study, weak alignment of the second and third transmembrane domains (TM23) of the human glycine receptor (GlyR) was achieved in low-q bicelles (q = DMPC/DHPC). Although protein-free bicelles with such low q would likely show isotropic properties, the insertion of TM23 induced weakly preferred orientations so that the RDC of the embedded protein can be measured. The extent of the alignment increases but the TM23 signal intensity decreases when q varied from 0.19 to 0.60. A q of 0.50 was found to be an optimal compromise between alignment and the signal-to-noise ratio. In each pair of NMR experiments for RDC measurements, the same sample and pulse sequence were used, with one being performed at high-resolution magic-angle spinning to obtain pure J-couplings without RDC. A meaningful structure refinement in bicelles was possible by iteratively fitting of the experimental RDCs to the back-calculated RDCs using the high-resolution NMR structure of GlyR TM23 in trifluoroethanol as the starting template. The combination of this method with the conventional high-resolution NMR in the membrane-mimicking solvent-water mixtures offers an attractive way to derive structural information for membrane proteins in their native environment.