Protein Folding Cooperativity and Thermodynamic Barriers of the Simplest β-Sheet Fold: A Survey of WW Domains

Theory and experiments have shown that microsecond folding proteins exhibit characteristic thermodynamic properties that reflect the limited cooperativity of folding over marginal barriers (downhill folding). Those studies have mostly focused on proteins with large alpha-helical contents and small size, which tend to be the fastest folders. A key open question is whether such properties are also present in the fastest all-beta proteins. We address this issue by investigating the unfolding thermodynamics of a collection of WW domains as representatives of the simplest beta-sheet fold. WW domains are small microsecond folders, although they do not fold as fast as their alpha-helical counterparts. In previous work on the NEDD4-WW4 domain, we reported deviations thermodynamics that were less apparent and thus suggestive of an incipient downhill scenario. Here we investigate the unfolding thermodynamics of four other WW domains (NEDD4-WW3, YAP65-WW1(L30K), FBP11-WW1, and FBP11WW2) by performing all of the thermodynamic tests for downhill folding that have been previously developed on alpha-helical proteins. This set of five WAN domains shares low sequence identity and include examples from two specificity classes, thus providing a comprehensive survey. Thermodynamic analysis of the four new WW domains consistently reveals all of the properties of downhill folding equilibria, which are in all cases more marked than what we found before in NEDD4-WW4. Our results show that fast-folding all-beta proteins do share limited cooperativity and gradual unfolding thermodynamics with fast alpha-helical proteins and suggest that the free energy barrier to folding of natural proteins is mostly determined by size and fold topology and much less by the specific amino acid sequence.