title: Slow proton transfer coupled to unfolding explains the puzzling results of single-molecule experiments on BBL, a paradigmatic downhill folding protein.
abstract: A battery of thermodynamic, kinetic, and structural approaches has indicated that the small α-helical protein BBL folds-unfolds via the one-state downhill scenario. Yet, single-molecule fluorescence spectroscopy offers a more conflicting view. Single-molecule experiments at pH 6 show a unique half-unfolded conformational ensemble at mid denaturation, whereas other experiments performed at higher pH show a bimodal distribution, as expected for two-state folding. Here we use thermodynamic and laser T-jump kinetic experiments combined with theoretical modeling to investigate the pH dependence of BBL stability, folding kinetics and mechanism within the pH 6-11 range. We find that BBL unfolding is tightly coupled to the protonation of one of its residues with an apparent pKa of ~ 7. Therefore, in chemical denaturation experiments around neutral pH BBL unfolds gradually, and also converts in binary fashion to the protonated species. Moreover, under the single-molecule experimental conditions (denaturant midpoint and 279 K), we observe that proton transfer is much slower than the ~ 15 microseconds folding-unfolding kinetics of BBL. The relaxation kinetics is distinctly biphasic, and the overall relaxation time (i.e. 0.2-0.5 ms) becomes controlled by the proton transfer step. We then show that a simple theoretical model of protein folding coupled to proton transfer explains quantitatively all these results as well as the two sets of single-molecule experiments, including their more puzzling features. Interestingly, this analysis suggests that BBL unfolds following a one-state downhill folding mechanism at all conditions. Accordingly, the source of the bimodal distributions observed during denaturation at pH 7-8 is the splitting of the unique conformational ensemble of BBL onto two slowly inter-converting protonation species. Both, the unprotonated and protonated species unfold gradually (one-state downhill), but they exhibit different degree of unfolding at any given condition because the native structure is less stable for the protonated form.
authors.affiliation: Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Madrid, Spain ; IMDEA-Nanociencia, Madrid, Spain.
authors.forename: Michele
authors.initials: M
authors.lastname: Cerminara
authors.forename: Luis A
authors.initials: LA
authors.lastname: Campos
authors.forename: Ravishankar
authors.initials: R
authors.lastname: Ramanathan
authors.forename: Victor
authors.initials: V
authors.lastname: Muñoz
mesh_terms: D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D017510:Protein Folding; D055550:Protein Stability; D011506:Proteins / Q000737:chemistry*; D011522:Protons
chemical_list: D011506:Proteins; D011522:Protons