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pK_a Values for Side-Chain Carboxyl Groups of a PGB1 Variant Explain Salt and pH-Dependent Stability

Stina Lindman ^*, Sara Linse ^*, Frans A. A. Mulder  and Ingemar André ^*

^* Department of Biophysical Chemistry, Lund University, Chemical Center, SE-22100 Lund, Sweden; and Biophysical Chemistry, Groningen University, Nijenborgh 4, 9747 AG Groningen, The Netherlands

Correspondence: Address reprint requests to Stina Lindman or Sara Linse, Lund University, Chemical Center, SE-22100 Lund, Sweden. Tel.: 46-46-222-7092; Fax: 46-46-222-4543; E-mails: Stina.Lindman{at}bpc.lu.se, Sara.Linse{at}bpc.lu.se.

Determination of pK_a values of titrating residues in proteins provides a direct means of studying electrostatic coupling as well as pH-dependent stability. The B1 domain of protein G provides an excellent model system for such investigations. In this work, we analyze the observed pK_a values of all carboxyl groups in a variant of PGB1 (T2Q, N8D, N37D) at low and high ionic strength as determined using ¹H-¹³C heteronuclear NMR in a structural context. The pK_a values are used to calculate the pH-dependent stability in low and high salt and to investigate electrostatic coupling in the system. The observed pK_a values can explain the pH dependence of protein stability but require pK_a shifts relative to model values in the unfolded state, consistent with persistent residual structure in the denatured state. In particular, we find that most of the deviations from the expected random coil values can be explained by a significantly upshifted pK_a value. We show also that ¹³C backbone carbonyl data can be used to study electrostatic coupling in proteins and provide specific information on hydrogen bonding and electrostatic potential at nontitrating sites.