Heterogeneous folding of the trpzip hairpin: full atom simulation and experiment.

The beta-hairpin trpzip2 can be tuned continuously from a two-state folder to folding on a rough energy landscape without a dominant refolding barrier. At high denaturant concentration, this extremely stable peptide exhibits a single apparent "two-state" transition temperature when monitored by different spectroscopic techniques. However, under optimal folding conditions the hairpin undergoes an unusual folding process with three clusters of melting transitions ranging from 15 degrees C to 160 degrees C, as monitored by 12 different experimental and computational observables. We explain this behavior in terms of a rough free energy landscape of the unfolded peptide caused by multiple tryptophan interactions and alternative backbone conformations. The landscape is mapped out by potentials of mean force derived from replica-exchange molecular dynamics simulations. Implications for deducing cooperativity from denaturant titrations, for the origin of folding cooperativity, and for the folding of thermophilic proteins are pointed out. trpzip is an excellent small tunable model system for the glass-like folding transitions predicted by landscape theory.