| Cis/trans isomerization of proline plays a key role in protein folding and the conformational heterogeneity of biomolecules. As a result, proline oligomer is widely studied as a model system. Solvent influences the configurational preferences of polyproline; the all-cis right-handed polyproline helix (polyproline-I, PPI) is favored in 1-propanol, while the all-trans left-handed helix (polyproline-II, PPII) is present in water. This work focuses on exploring the structural transition of polyproline using hyphenated ion mobility (IM) spectrometry and mass spectrometry (MS) techniques. A thirteen-residue polyproline (polyproline-13, Pro13) is analyzed as the all-cis PPI helix is converted to the all- trans PPII helix. IM profiles as a function of transition time suggest that when PPI is introduced in water, it folds into PPII through at least six intermediates via a sequential mechanism. Kinetic and equilibrium measurements of this process, as well as for the reversed transition, were also performed at multiple temperatures to establish detailed energy surface for this system. It appears that early PPI → PPII intermediates are energetically favorable due to the hydration of the peptide backbone. Although late intermediates are enthalpically unfavorable, peptide bonds continue flipping because of an increase in system entropy. Upon immersion of hydrated-PPII in 1-propanol, the reverse transition is induced and proceeds through a parallel mechanism involving multiple pathways that are driven by entropy. In addition, a two-dimensional IM (IM-IM-MS) technique was utilized to select and activate eight specific conformations that are found during the PPI → PPII transition of Pro13 in water. Upon gentle collisional activation, each selected solution-phase conformer appears to refold into the all-cis PPI helix in vacuo. Further analysis shows that in the absence of solvent, the formation of the PPI helix occurs via similar parallel pathways discovered for the PPII → PPI transition in 1-propanol. Finally, when polyproline-7 was transferred from 1-propanol into aqueous environment under acidic conditions, a remarkably-slow protonation reaction coupled with the PPI → PPII conversion was observed. Kinetics and equilibrium measurements as a function of temperature allow determination of the thermochemistry and insight into how proton transfer is regulated in this system. |
