Downhill versus Barrier-Limited Folding of BBL 1: Energetic and Structural Perturbation Effects upon Protonation of a Histidine of Unusually Low pKa

A dispersion of melting temperatures at pH5.3 for individual I residues of the BBL protein domain has been adduced as evidence for barrier-free downhill folding. Other members of the peripheral Subunit domain family fold cooperatively at pH7. To search for possible causes of anomalies in BBL's denaturation behavior, we measured the pH titration of individual residues by heteronuclear NMR. At 298 K, the pK(a) of His142 was close to that of free histidine at 6.47 +/- 0.04, while that of the more buried His166 was highly perturbed at 5.39 +/- 0.02. Protonation of His166 is thus energetically unfavorable and destabilizes the protein by similar to 1.5 kcal/mol. Changes in C-alpha secondary shifts at pH5.3 showed a decrease in helicity of the C-terminus of helix 2, where His-166 is located, which was accompanied by a measured decrease of 1.1 +/- 0.2 kcal/mol in stability from pH 7 to 5.3. Protonation of His166 perturbs, therefore, the structure of BBL. Only similar to 1% of the structurally perturbed state will be present at the biologically relevant pH7.6. Experiments at pH5.3 report on a near-equal mixture of the two different native states. Further, at this pH, small changes of pH and pK(a) induced by changes in temperature will have near-maximal effects on pH-dependent conformational equilibria and on propagation of experimental error. Accordingly, conventional barrier-limited folding predicts some dispersion of measured thermal unfolding curves of individual residues at pH 5.3. (C) 2008 Elsevier Ltd. All rights reserved.