pH-Dependent Cooperativity and Existence of a Dry Molten Globule in the Folding of a Miniprotein BBL

Solution pH plays an important role in protein dynamics, stability, and folding; however, detailed mechanisms remain poorly understood. Here we use continuous constant pH molecular dynamics in explicit solvent with pH replica exchange to explore the pH-dependent stability and folding mechanism of a miniprotein BBL, which has drawn intense debate in the past. Consistent with the two-state model, simulations showed native and denatured states with pH-dependent populations. However, at pH 7, the folding barrier is marginal and it vanishes as pH is decreased to 5, in agreement with the downhill folding hypothesis. As pH continues to decrease, the unfolding barrier lowers and denaturation is triggered by the protonation of Asp162, consistent with experimental evidence. Interestingly, unfolding proceeded via a sparsely populated intermediate, with intact secondary structure and a compact, unlocked hydrophobic core shielded from solvent, lending support to the recent hypothesis of a universal dry molten globule in protein folding. Our work demonstrates that constant pH molecular dynamics is a unique tool for testing this and other hypotheses to advance the knowledge in protein dynamics, stability, and folding.