 protein folding cooperativity is defined by the nature of the finite - size thermodynamic transition exhibited upon folding : two - state transitions show a free - energy barrier between the folded and unfolded ensembles , while downhill folding is barrierless . a microcanonical analysis , 
 where the energy is the natural variable , has proved to be better suited than its canonical counterpart to unambiguously characterize the nature of the transition . 
 replica - exchange molecular dynamics simulations of a high - resolution coarse - grained model allow for the accurate evaluation of the density of states in order to extract precise thermodynamic information and measure its impact on structural features . 
 the method has been applied to three helical peptides : a short helix shows sharp features of a two - state folder , while a longer helix and a three - helix bundle exhibit downhill and two - state transitions , respectively . extending the results of lattice simulations and theoretical models , we have found that it is the interplay between secondary structure and the loss of non - native tertiary contacts that determines the nature of the transition . 
Supplementary Material