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 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 approximately 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 His166 is located, which was accompanied by a measured decrease of 1.1+/-0.2 kcal/mol in stability from pH7 to 5.3. Protonation of His166 perturbs, therefore, the structure of BBL. Only approximately 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 pH5.3.