pK(a) Values for the Unfolded State under Native Conditions Explain the pH-Dependent Stability of PGB1.

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pK(a) Values for the Unfolded State under Native Conditions Explain the pH-Dependent Stability of PGB1. / Lindman, Stina; Bauer, Mikael; Lund, Mikael; Diehl, Carl; Mulder, Frans; Akke, Mikael; Linse, Sara.

In: Biophysical Journal, Vol. 99, No. 10, 2010, p. 3365-3373.

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T1 - pK(a) Values for the Unfolded State under Native Conditions Explain the pH-Dependent Stability of PGB1.

AU - Lindman, Stina

AU - Bauer, Mikael

AU - Lund, Mikael

AU - Diehl, Carl

AU - Mulder, Frans

AU - Akke, Mikael

AU - Linse, Sara

N1 - The information about affiliations in this record was updated in December 2015. The record was previously connected to the following departments: Biophysical Chemistry (LTH) (011001011), Theoretical Chemistry (S) (011001039), Biochemistry and Structural Biology (S) (000006142)

PY - 2010

Y1 - 2010

N2 - 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.

AB - 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.

U2 - 10.1016/j.bpj.2010.08.078

DO - 10.1016/j.bpj.2010.08.078

M3 - Article

VL - 99

SP - 3365

EP - 3373

JO - Biophysical Journal

JF - Biophysical Journal

SN - 1542-0086

IS - 10

ER -