Characterisation of calmodulin structural transitions by ion mobility mass spectrometry

Abstract

This study demonstrates the ability of travelling wave ion mobility-mass spectrometry to measure collision cross-sections of ions in the negative mode, using a calibration based approach. Here, negative mode ion mobility-mass spectrometry was utilised to understand structural transitions of calmodulin upon Ca2þ binding and complexation with model peptides melittin and the plasma membrane Ca2þ pump C20W peptide. Coexisting calmodulin conformers were distinguished on the basis of their mass and cross-section, and identified as relatively folded and unfolded populations, with good agreement in collision cross-section to known calmodulin geometries. Titration of calcium tartrate to physiologically relevant Ca2þ levels provided evidence for intermediately metalated species during the transition from apo- to holo-calmodulin, with collision cross-section measurements indicating that higher Ca2þ occupancy is correlated with more compact structures. The binding of two representative peptides which exemplify canonical compact (melittin) and extended (C20W) peptidecalmodulin binding models has also been interrogated by ion mobility mass spectrometry. Peptide binding to calmodulin involves intermediates with metalation states from 1–4 Ca2þ, which demonstrate relatively collapsed structures, suggesting neither the existence of holo-calmodulin or a pre-folded calmodulin conformation is a prerequisite for binding target peptides or proteins. The biological importance of the different metal unsaturated calmodulin complexes, if any, is yet to be understood.