Microscopic effects of proline co-solvent on alanine homopeptide structure, solvation and helix folding dynamics

We present a computational investigation to explore the influence of the protective osmolyte proline as a co-solvent on peptide structure and dynamics for a series of alanine-based peptides, (ALA)n of length n = 5, 8, 15, and 21 residues. Applying multi-microsecond molecular dynamics simulations in a 2 M proline solution, we evaluate peptide structure, solvation and helix folding dynamics and compare to behavior in pure water. Proline addition enhances helix content and significantly slows folding and unfolding times, correlating with a 1.9-fold increase in solvent viscosity. Notably, ALA15 helix content increases from 25% to 49% and relaxation time rises from 110 ns to 540 ns in proline relative to water. Microscopic solvation effects of proline include peptide compaction and dehydration, exclusion of proline from the backbone, formation of weak interactions with the ALA methyl sidechains, and strong interactions with water. The differences of these effects on the helix and coil states drive helix stabilization by proline. Low-dimensional kinetic modeling with Optimal Dimensionality Reduction predicts distinct folding mechanisms: shorter peptides (ALA5-ALA15) exhibit direct helix-coil transitions, and only the longest ALA21 follows a more complex folding pathway involving intermediates. Statistically, enhanced stability of hydrogen bonds in the peptide centers and strong correlation between transitions on neighboring residues are shared between water and proline solutions. However, there is a preference for helix initiation at the N-terminus under proline influence. Our analysis describes the molecular mechanisms of how proline modulates peptide behavior, offering atomistic insights into helix stabilization and folding mechanisms mediated by osmolytes.