SAXS determination of the conformational information of VPPase embedded in a phospholipid nanodisc environment

Orion Shih^1*, Yi-Qi Yeh¹, Kuei-Fen Liao¹, Yun-Hsuan Kuo², Richard K. Heenan⁵, Yun-Wei Chiang², Yuh-Ju Sun⁴, U-Ser Jeng^1,3

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, email:shih.orion@nsrrc.org.tw

Vigna radiata H⁺-PPase (VPPase) is a proton pump that hydrolyzes pyrophosphate (PP_i) 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 PP_i-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, PP_i-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