Reframing the Protein Folding Problem: Entropy as Organizer.

It has been a long-standing conviction that a protein's native fold is selected from a vast number of conformers by the optimal constellation of enthalpically favorable interactions. In marked contrast, this Perspective introduces a different mechanism, one that emphasizes conformational entropy as the principal organizer in protein folding while proposing that the conventional view is incomplete. This mechanism stems from the realization that hydrogen bond satisfaction is a thermodynamic necessity. In particular, a backbone hydrogen bond may add little to the stability of the native state, but a completely unsatisfied backbone hydrogen bond would be dramatically destabilizing, shifting the U(nfolded) ⇌ N(ative) equilibrium far to the left. If even a single backbone polar group is satisfied by solvent when unfolded but buried and unsatisfied when folded, that energy penalty alone, approximately +5 kcal/mol, would rival almost the entire free energy of protein stabilization, typically between -5 and -15 kcal/mol under physiological conditions. Consequently, upon folding, buried backbone polar groups must form hydrogen bonds, and they do so by assembling scaffolds of α-helices and/or strands of β-sheet, the only conformers in which, with rare exception, hydrogen bond donors and acceptors are exactly balanced. In addition, only a few thousand viable scaffold topologies are possible for a typical protein domain. This thermodynamic imperative winnows the folding population by culling conformers with unsatisfied hydrogen bonds, thereby reducing the entropy cost of folding. Importantly, conformational restrictions imposed by backbone···backbone hydrogen bonding in the scaffold are sequence-independent, enabling mutation─and thus evolution─without sacrificing the structure.