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

Protein complexes are essential for proteins' folding and biological function. Currently, native analysis of large multimeric protein complexes remains challenging. Structural biology techniques are time-consuming and often cannot monitor the proteins' dynamics in solution. Here, a capillary electrophoresis-mass spectrometry (CE-MS) method is reported to characterize, under near-physiological conditions, the conformational rearrangements of ∽1 MDa GroEL upon complexation with binding partners involved in a protein folding cycle. The developed CE-MS method is fast (30 min per run), highly sensitive (low-amol level), and requires ∽10 000-fold fewer samples compared to biochemical/biophysical techniques. The method successfully separates GroEL₁₄ (∽800 kDa), GroEL₇ (∽400 kDa), GroES₇ (∽73 kDa), and NanA₄ (∽130 kDa) oligomers. The non-covalent binding of natural substrate proteins with GroEL₁₄ can be detected and quantified. The technique allows monitoring of GroEL₁₄ conformational changes upon complexation with (ATPγS)_4-14 and GroES₇ (∽876 kDa). Native CE-pseudo-MS³ analyses of wild-type (WT) GroEL and two GroEL mutants result in 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 direct infusion ESI-MS. This study shows the CE-MS potential to provide information on binding stoichiometry and kinetics for various protein complexes.