An Experimental Study of the Solvent-Dependent Self-Assembly/Disassembly and Conformer Preferences of Gramicidin A

The solvent dependence of self-assembly/disassembly kinetics and conformer preferences of the gramicidin A (GA) dimer is investigated using a combination of techniques, viz., electrospray ionization–ion mobility–mass spectrometry (IM-MS), collision-induced dissociation (CID), and hydrogen/deuterium exchange (HDX)-MS. IM-MS measurements reveal that there are possibly three distinct GA dimeric species, detected as sodium ion adduct ions [2GA + 2Na]²⁺, and these are assigned as the parallel β-helix, antiparallel β-helix, and head-to-head dimer. The monomerization kinetics and equilibrium abundances of the dimer ions depend upon solvent polarity. The antiparallel β-helix was the thermodynamically preferred species in less polar solvents. HDX measurements and collision-induced dissociation (CID) of the intermediate complex confirm the well-protected dimer geometry with strong intermolecular hydrogen bonds. This combined IM-HDX-CID methodology provides a comprehensive view of GA self-assembly/disassembly in low dielectric solutions, showing its potential utility in solving solution-phase protein self-assembly/disassembly kinetics and providing structural information of the multimers at the same time.