Resolving Surfactant Monormer- and Micelle-unfolding Stages of Bovine Serum Albumin Using Simultaneous HPLC/SAXS, UV-Vis Absorption, and Refractive Index.

Yi-Qi Yeh^1*, Kuei-Fen Liao¹, Orion Shih¹, Wei-Ru Wu¹, Chun-Jen Su¹, U-Ser Jeng^1,2

¹Scientific Research Group, National Synchrotron Radiation Research Center, Hsinchu, Taiwan
²Department of Chemical Engineering, National Tsing Hua University, Hsinchu, Taiwan

* Presenter:Yi-Qi Yeh, email:yeh.yq@nsrrc.org.tw

Detergents are commonly used to disrupt noncovalent interactions of proteins, leading to detergent-protein complexes or stabilized recombinant proteins. To understand their interactions, polarity, and stability in varied detergent concentrations, many methods have been used to investigate conformational changes of proteins and protein-detergent complexes. Spectroscopies could resolve the local structure of such protein/detergent complex. The MD simulation could propose the molecular mechanism by which detergent induces protein unfolding. However, resolving the corresponding stoichiometry and conformation of protein/detergent during unfolding requires separating the coexisting protein/detergent complex and SDS micelles in the solution.
HPLC/SAXS/UV-Vis/RI system (online High-Performance Liquid Chromatography/Small-angle X-ray scattering/UV-Vis/Refractive Index) in TPS 13A at NSRRC can quantitatively and qualitatively determine bio-macromolecule conformation the binding ratio of protein/detergent complex, and unfolding pathway of protein. In this work, we show that sodium dodecyl sulfate (SDS), a frequently used surfactant in the purification of membrane proteins, can bind to bovine serum albumin (BSA) for multistage unfolding. The integrated system can separate the scattering contributions from the BSA/SDS complexes and SDS micelles. SAXS/WAXS data were collected simultaneously and successively during the HPLC sample elution with 1 data frame per 2 s using vacuum Eiger-9M and 1MX detectors. Together with integrated observations of UV-vis absorption and refractive index (RI), we have resolved the stoichiometric unfolding conformations of BSA by SDS monomers to micelles. The corresponding protein-SDS association numbers and Rg along the unfolding process are determined uniquely from a combined analysis of UV-Vis absorption, refractive index, and SAXS profile measured in one sample elution. The SDS/BSA binding ratios extracted from HPLC/MALS data were consistent with those observed from HPLC/SAXS/UV-Vis/RI data. It revealed that the SDS binding to albumin is a four-stage character involving monomer absorption, nucleation stage, and micelle growth.

Keywords: SAXS, BSA, unfolding, micelle