pKa-values for side-chain carboxyl groups of a PGB1 variant explain salt and pH dependent stability
Stina Lindman 1*, Sara Linse 1, Frans A. A. Mulder 2 and Ingemar André 3
1 Lund University
2 Groningen University
3 University of Washington
* To whom correspondence should be addressed. E-mail: stina.lindman{at}bpc.lu.se.
Submitted on May 9, 2006
Revised on July 3, 2006
Accepted on 21 September 2006
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Abstract |
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Determination of pKa 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 (PGB1) provides an excellent model system for such investigations. In this work we analyze the observed pKa 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 pKa values are used to calculate the pH dependent stability in low and high salt and to investigate electrostatic coupling in the system. The observed pKa values can explain the pH dependence of protein stability but require pKa 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 carboxyl group. 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 non-titrating sites.
Key Words:
Gaussian chain, Protein G B1, electrostatic interactions, pH dependent stability, pKa, protein stability
