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

Eyal Arbely, Trevor J. Rutherford, Timothy D. Sharpe, Neil Ferguson, Alan R. Fersht

Research output: Contribution to journalArticlepeer-review

26 Scopus citations

Abstract

A dispersion of melting temperatures at pH 5.3 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 pH 7. 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 pKa 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 ∼ 1.5 kcal/mol. Changes in Cα secondary shifts at pH 5.3 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 pH 7 to 5.3. Protonation of His166 perturbs, therefore, the structure of BBL. Only ∼ 1% of the structurally perturbed state will be present at the biologically relevant pH 7.6. Experiments at pH 5.3 report on a near-equal mixture of the two different native states. Further, at this pH, small changes of pH and pKa 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.

Original languageEnglish
Pages (from-to)986-992
Number of pages7
JournalJournal of Molecular Biology
Volume387
Issue number4
DOIs
StatePublished - 10 Apr 2009
Externally publishedYes

Keywords

  • NMR
  • downhill
  • histidine
  • perturbed pK
  • protein folding

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