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.

pH-Dependent cooperativity and existence of a dry molten globule in the folding of a miniprotein BBL

“…Note, while our data highlights the role of Asp162, it does not rule out the contribution from other acidic residues because their protonation increases the repulsive Coulombic forces due to the excess positive charges. Cerminara et al recently suggested that BBL unfolding could be due to the titration of an arginine 59 . Our simulations demonstrated that Lys and Arg residues are fully charged in the simulation pH range (1–7.5), thus ruling out their involvement in the unfolding of BBL.…”

Anesthetic Diffusion Through Lipid Membranes Depends on the Protonation Rate

“…Proton transfer plays an essential role in many biological systems 53 54 55 56 . Some reports have shown proton transfer rates in the order of femtoseconds - microseconds, highly depending on the chemical structure of the target molecule and its environment 57 58 59 60 . Recently, it has been reported slight but significant differences in the proton transfer rates in the lysosome region between normal lung cells (30 ps) and lung cancer cells (25 ps) 61 .…”

pH dependent protein stability: A quantitative approach based on Kramer's barrier escape

“…In a recent single molecule experimental study, Munoz et al [2] have observed the slow protonation coupled unfolding of a small ˛-helical BBL protein. On the basis of their observations, they resolved a puzzling fact: even though unfolding of BBL protein at pH = 6 is guided by a unimodal unique conformation, the unfolding process is followed with a bimodal distribution around pH = 7-8.…”

“…On the basis of their observations, they resolved a puzzling fact: even though unfolding of BBL protein at pH = 6 is guided by a unimodal unique conformation, the unfolding process is followed with a bimodal distribution around pH = 7-8. Munoz et al [2] have argued that at all conditions, BBL protein will have a barrier-less unfolding event, and thus, the unfolding is expected to be associated with a unimodal distribution. However, at around pH = 7-8, the protein will have another slow conformational change to its protonated species and that will be responsible for the bimodal distribution.…”

“…Thus a change in the folding kinetics due to the influence of any other chemical agent or ions is an important aspect of the folding mechanism. Many of the protein folding-unfolding kinetics are found to be triggered by Ca 2+ , H + or other ion concentrations [1][2][3][4]. These ions are found to play an important role in maintaining a well regulated physiologically viable timescale of the cell machinery by modulating the rate of the folding-unfolding kinetics of the involved proteins.…”