Retention and Rearrangement of Membrane Protein Complexes' Higher Order Structure by Collisionally Activated Dissociation- and Electron Capture Dissociation-Mass Spectrometry.

Abstract

Membrane proteins (MPs) challenge biophysical and structural biology methods. Native mass spectrometry (MS) has emerged as a powerful tool to study MP structures and their modulation by lipids. We examine the pathways by which three MPs, tetrameric Aquaporin Z (AqpZ), trimeric ammonium transporter (AmtB), and pentameric mechanosensitive channel of large conductance (MscL) decompose following collisionally activated dissociation (CAD) or electron capture dissociation (ECD) in native top-down MS. MPs subjected to CAD typically decompose along well-characterized pathways releasing highly charged monomers and low-charge state complementary subunits, but a lesser pathway accessed by low pressure collisions cleaves the backbone into fragments covering much of the sequence. Collisions also rearrange structures, e.g., AmtB subunits rearrange to form novel interactions and/or salt bridges that surprisingly retain a formerly surface-exposed segment despite ejecting the binding interface. MscL dissociation pathways depend on precursor charge-state, a behavior observed in a few soluble complexes, yet unaddressed mechanistically. Salt bridges in low charge state complexes stabilize subunits from ejection while facilitating only smaller, local rearrangements that release covalently cleaved products from transmembrane regions. With fewer opposite charges, on average, higher charge state molecules can rearrange intersubunit salt bridges on the experimental time scale to partition charge asymmetrically and free a subunit. ECD with supplemental activation can retain higher order structures of proteins and inform about the strongly interacting regions that preclude product ion release. With extensive regions lacking ionizable residues, MPs enable key interactions that guide the structure and dynamics of gas phase protein assemblies to be probed.