The effects of proline co-solvent on helix folding are explored through the single discrete coordinate of the number of helical hydrogen bonds. The analysis is based on multi-microsecond length molecular dynamics simulations of alanine-based helix-forming peptides, (ALA)n, of length n = 4, 8, 15 and 21 residues, in an aqueous solution with 2 M concentration of proline. The effects of addition of proline on the free energy landscape for helix folding were analyzed using the graph-based Dijkstra algorithm, Optimal Dimensionality Reduction kinetic coarse graining, committor functions, as well as through the diffusion of the helix boundary. Viewed at a sufficiently long time-scale, helix folding in the coarse-grained hydrogen bond space follows a consecutive mechanism, with well-defined initiation and propagation phases, and an interesting set of intermediates. Proline addition slows down the folding relaxation of all four peptides, increases helix content and induces subtle mechanistic changes compared to pure water solvation. A general trend is for transition state shift towards earlier stages of folding in proline relative to water. For ALA5 and ALA8 direct folding is dominant. In ALA8 and ALA15 multiple pathways appear possible. For ALA21 a simple mechanism emerges, with a single path from helix to coil through a set of intermediates. Overall, this work provides new insights into effects of proline co-solvent on helix folding, complementary to more standard approaches based on three-dimensional molecular structures. [ABSTRACT FROM AUTHOR]

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