title: pK(a) values for the unfolded state under native conditions explain the pH-dependent stability of PGB1.
abstract: 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 pK(a) values. However, pK(a) values of the unfolded state are rarely accessible under native conditions, where the unfolded state has a very low population. Here, we report pK(a) values under nondenaturing conditions for two unfolded fragments of the protein G B1 domain that mimic the unfolded state of the intact protein. pK(a) values were determined for carboxyl groups by monitoring their pH-dependent (13)C 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 pK(a) 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 pK(a) values of the folded and unfolded states and compared to experimental stability data. The use of experimental pK(a) values for the unfolded state results in significantly improved agreement with experimental data, as compared to calculations based on model data alone.
authors.affiliation: Center for Molecular Protein Science, Lund University, Lund, Sweden.
authors.forename: Stina
authors.initials: S
authors.lastname: Lindman
authors.forename: Mikael C
authors.initials: MC
authors.lastname: Bauer
authors.forename: Mikael
authors.initials: M
authors.lastname: Lund
authors.forename: Carl
authors.initials: C
authors.lastname: Diehl
authors.forename: Frans A A
authors.initials: FA
authors.lastname: Mulder
authors.forename: Mikael
authors.initials: M
authors.lastname: Akke
authors.forename: Sara
authors.initials: S
authors.lastname: Linse
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D011522:Protons; D013816:Thermodynamics; D017186:Titrimetry
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D050505:Mutant Proteins; D011522:Protons