Stoichiometric unfolding of bovine serum albumin by surfactant, as revealed from HPLC/SAXS with online observation of 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

¹National 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 complex or stabilized recombinant proteins. In past, many methods have been used to investigate conformational changes of proteins and protein-detergent complexes to understand their interactions, polarity and stability in varied detergent concentrations. The local structure of such protein/detergent complex could be resolved by spectroscopies; however, resolving the corresponding stoichiometric protein unfolding conformation requires separating the effects contributed by the coexisted protein/detergent complex and SDS micelles in the solution.
In this work, we show that sodium dodecyl sulfate (SDS), a frequently used surfactant in purification of membrane proteins, can bind to bovine serum albumin (BSA) for multistage unfolding. The on-line protein purification system of high performance liquid chromatography (SEC-HPLC) incorporated to the synchrotron small-angle X-ray scattering (SAXS) instrument of the TPS 13A BioSAXS at NSRRC, allows separating the scattering contributions from the BSA/SDS complexes and SDS micelles. SAXS/WAXS data were simultaneously and successively collected during the HPLC sample elution with 1 data frame per 2 s using in 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 along the unfolding process are determined uniquely from a combined analysis of UV-Vis absorption, refractive index, and zero-angle SAXS intensity measured in one sample elution.

Keywords: BioSAXS, protein unfolding, BSA