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 similar to 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 similar to 10 000-foldfewer samples compared to biochemical/biophysical techniques. The methodsuccessfully separates GroEL14 (similar to 800 kDa), GroEL7 (similar to 400 kDa), GroES7 (similar to 73 kDa), and NanA4 (similar to 130 kDa) oligomers. The non-covalent binding of naturalsubstrate proteins with GroEL14 can be detected and quantified.The technique allows monitoring of GroEL14 conformational changesupon complexation with (ATP gamma S)4-14 and GroES7 (similar to 876 kDa). NativeCE-pseudo-MS3 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. High-sensitivity capillary electrophoresis-mass spectrometry (CE-MS) method to quickly characterize approximate to 1 MDa GroEL14 multimeric protein assembly under near-physiological conditions is presented. The CE-MS method enables assessment of non-covalent binding of substrate proteins to GroEL14 chaperon and its conformational changes upon complexation with nucleotides and GroES7 involved in a protein folding cycle, and in-depth structural characterization of wild-type and mutant GroEL14 complexes.image