  Two-state cooperativity is an important characteristic in protein folding. It
is defined by a depletion of states lying energetically between folded and
unfolded conformations. While there are different ways to test for two-state
cooperativity, most of them probe indirect proxies of this depletion. Yet,
generalized-ensemble computer simulations allow to unambiguously identify this
transition by a microcanonical analysis on the basis of the density of states.
Here we perform a detailed characterization of several helical peptides using
coarse-grained simulations. The level of resolution of the coarse-grained model
allows to study realistic structures ranging from small alpha-helices to a de
novo three-helix bundle - without biasing the force field toward the native
state of the protein. Linking thermodynamic and structural features shows that
while short alpha-helices exhibit two-state cooperativity, the type of
transition changes for longer chain lengths because the chain forms multiple
helix nucleation sites, stabilizing a significant population of intermediate
states. The helix bundle exhibits the signs of two-state cooperativity owing to
favorable helix-helix interactions, as predicted from theoretical models. The
detailed analysis of secondary and tertiary structure formation fits well into
the framework of several folding mechanisms and confirms features observed so
far only in lattice models.
