SAXS determination of the conformational information of VPPase embedded in a phospholipid nanodisc environment
Orion Shih1*, Yi-Qi Yeh1, Kuei-Fen Liao1, Yun-Hsuan Kuo2, Richard K. Heenan5, Yun-Wei Chiang2, Yuh-Ju Sun4, U-Ser Jeng1,3
1National Synchrotron Radiation Research Center, Hsinchu, Taiwan
2Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan
3Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan
4Department of Life Science and Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan
5STFC ISIS Facility, Rutherford-Appleton Laboratory, Didcot, UK
* Presenter:Orion Shih,
Vigna radiata H+-PPase (VPPase) is a proton pump that hydrolyzes pyrophosphate (PPi) to drive proton transportation across cellular membranes against the electrochemical gradient. The detailed mechanisms underlying the translocation reactions and structural changes between different conformational states of VPPase (ligand-free or PPi-binding) are unclear. In this report, high-performance-liquid-chromatography, small-angle X-ray scattering (SAXS), UV−Vis absorption, differential refractive index (RI) detections, and modified core-shell bicelle model fitting are integrated to probe the structural information of VPPase-incorporated POPC nanodiscs. The results indicate that the hydrophobic region of VPPase thickens and overall structural fixedness increases when changing from resting state (R-state, ligand free) to initiated state (I-state, PPi-binding). This integrated analysis scheme can be applied onto other membrane protein/detergents/lipids complex and provides a new approach to membrane protein studies.

Keywords: Pyrophosphatase, SAXS, nanodisc, membrane protein