Native Capillary Electrophoresis-Mass Spectrometry of Near 1 MDa Non-Covalent GroEL/GroES/Substrate Protein Complexes.

Protein complexes are essential forproteins' folding and biological function. Currently, native analysis of largemultimeric protein complexes remains challenging. Structural biology techniquesare time-consuming and often cannot monitor the proteins' dynamics in solution. Here, a capillary electrophoresis-mass spectrometry (CE-MS) method is reportedto characterize, under near-physiological conditions, the conformationalrearrangements of ∽1 MDa GroEL upon complexation with binding partners involvedin a protein folding cycle. The developed CE-MS method is fast (30min per run), highly sensitive (low-amol level), and requires ∽10 000-foldfewer samples compared to biochemical/biophysical techniques. The methodsuccessfully separates GroEL 14 (∽800 kDa), GroEL7 (∽400 kDa), GroES 7 (∽73 kDa), and NanA 4 (∽130 kDa) oligomers. The non-covalent binding of naturalsubstrate proteins with GroEL 14 can be detected and quantified.The technique allows monitoring of GroEL 14 conformational changesupon complexation with (ATPγS) 4-14 and GroES 7 (∽876 kDa). NativeCE-pseudo-MS 3 analyses of wild-type (WT) GroEL and two GroEL mutants resultin up to 60% sequence coverage and highlight subtle structural differences between WT and mutated GroEL. The presented results demonstrate the superior CE-MS performance for multimeric complexes' characterization versus directinfusion ESI-MS. This study shows the CE-MS potential to provide information onbinding stoichiometry and kinetics for various protein complexes.