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PKa values for the unfolded state under native conditions explain the pH-dependent stability of PGB1

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  • Stina Lindman, Center for Molecular Protein Science, Unknown
  • Mikael C. Bauer, Center for Molecular Protein Science, Unknown
  • Mikael Lund, Lund University, Unknown
  • Carl Diehl, Center for Molecular Protein Science, Unknown
  • Frans A A Mulder
  • Mikael Akke, Center for Molecular Protein Science, Sweden
  • Sara Linse, Department of Biophysical Chemistry, Netherlands

Understanding the role of electrostatics in protein stability requires knowledge of these interactions in both the folded and unfolded states. Electrostatic interactions can be probed experimentally by characterizing ionization equilibria of titrating groups, parameterized as pKa values. However, pKa values of the unfolded state are rarely accessible under native conditions, where the unfolded state has a very low population. Here, we report pKa values under nondenaturing conditions for two unfolded fragments of the protein G B1 domain that mimic the unfolded state of the intact protein. pKa values were determined for carboxyl groups by monitoring their pH-dependent 13C chemical shifts. Monte Carlo simulations using a Gaussian chain model provide corrections for changes in electrostatic interactions that arise from fragmentation of the protein. Most pKa values for the unfolded state agree well with model values, but some residues show significant perturbations that can be rationalized by local electrostatic interactions. The pH-dependent stability was calculated from the experimental pKa values of the folded and unfolded states and compared to experimental stability data. The use of experimental pKa values for the unfolded state results in significantly improved agreement with experimental data, as compared to calculations based on model data alone.

Original languageEnglish
JournalBiophysical Journal
Pages (from-to)3365-3373
Number of pages9
Publication statusPublished - 17 Nov 2010
Externally publishedYes

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