Unraveling ShuA detergent-induced colloidal behavior in solution: A comprehensive SEC-MALS, SAXS, and SANS study.

In this study, we investigate the detergent-induced behavior of the integral membrane protein ShuA in solution, focusing on its interactions with octyl polyoxyethylene (OPOE) and n-dodecyl-β-D-maltoside (DDM). Using a combination of size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) and small-angle scattering techniques (SAXS and SANS), we provide a detailed characterization of the protein-detergent complex (PDC) behavior under varying conditions. Our results reveal that ShuA remains monomeric in 1% OPOE, whereas in 0.5 mM DDM, it undergoes a reversible monomer/dimer equilibrium that shifts towards a monodisperse, monomeric state with increasing DDM concentration to 7.5 mM, highlighting the significant influence of detergent type and concentration on protein colloidal stability. These findings have direct implications for membrane protein purification and structural studies, particularly in crystallization and cryo-EM sample preparation. The study emphasizes the necessity of optimizing detergent conditions to ensure monodispersity and structural integrity, preventing detergent-induced artifacts that could affect structural interpretations. Importantly, our results highlight the power of the SEC-MALS technique in determining oligomeric or association equilibrium states, detecting weak intermolecular interactions often overlooked in conventional SEC, and achieving this even in the particularly complex case of MPs. By integrating advanced scattering techniques, this work contributes valuable insights into MP colloidal behavior, refining strategies for structural characterization and providing a framework for optimizing detergent conditions in biochemical and biophysical studies.