A method to determine residue-specific unfolded-state pKa values from analysis of stability changes in single mutant cycles.

It is now widely recognized that the unfolded state of a protein in equilibrium with the native state under folding conditions may contain significant residual structures. However, due to technical difficulties residue-specific interactions in the unfolded state remain elusive. Here we introduce a method derived from the Wyman-Tanford theory to determine residue-specific pK(a)'s in the unfolded state. This method requires equilibrium stability measurements of the wild type and single-point mutants in which titrable residues are replaced with charge-neutral ones under two pH conditions. Application of the proposed approach reveals a highly depressed pK(a) for Asp8 in the unfolded state of the NTL9 protein. Knowledge of unfolded-state pK(a)'s enables quantitative estimation of the unfolded-state electrostatic effects on protein stability. It also provides valuable benchmarks for the improvement of force fields and validation of microscopic information from molecular dynamics simulations.

[1]  Charles L Brooks,et al.  Exploring atomistic details of pH-dependent peptide folding , 2006, Proceedings of the National Academy of Sciences.

[2]  J. Wyman,et al.  LINKED FUNCTIONS AND RECIPROCAL EFFECTS IN HEMOGLOBIN: A SECOND LOOK. , 1964, Advances in protein chemistry.

[3]  Electrostatic effects in unfolded staphylococcal nuclease , 2008, Protein science : a publication of the Protein Society.

[4]  A. Fersht,et al.  Perturbed pKA-values in the denatured states of proteins. , 1995, Journal of molecular biology.

[5]  H. Bosshard,et al.  Inverse electrostatic effect: electrostatic repulsion in the unfolded state stabilizes a leucine zipper. , 2004, Biochemistry.

[6]  D. Raleigh,et al.  Mutational analysis demonstrates that specific electrostatic interactions can play a key role in the denatured state ensemble of proteins. , 2005, Journal of molecular biology.

[7]  Kevin L. Shaw,et al.  Charge–charge interactions influence the denatured state ensemble and contribute to protein stability , 2000, Protein science : a publication of the Protein Society.

[8]  I. André,et al.  pK(a) values for side-chain carboxyl groups of a PGB1 variant explain salt and pH-dependent stability. , 2007, Biophysical journal.

[9]  C. Tanford.  Protein denaturation. , 1968, Advances in protein chemistry.

[10]  B. Kuhlman,et al.  pKa values and the pH dependent stability of the N-terminal domain of L9 as probes of electrostatic interactions in the denatured state. Differentiation between local and nonlocal interactions. , 1999, Biochemistry.

[11]  D. Raleigh,et al.  Electrostatic interactions in the denatured state ensemble: their effect upon protein folding and protein stability. , 2008, Archives of biochemistry and biophysics.

[12]  V L Arcus,et al.  pKA values of carboxyl groups in the native and denatured states of barnase: the pKA values of the denatured state are on average 0.4 units lower than those of model compounds. , 1995, Biochemistry.

[13]  Steven T. Whitten and,et al.  pH dependence of stability of staphylococcal nuclease: evidence of substantial electrostatic interactions in the denatured state. , 2000 .

[14]  F A Quiocho,et al.  Carboxypeptidase A: a protein and an enzyme. , 1971, Advances in protein chemistry.

[15]  Sheena E Radford,et al.  An expanding arsenal of experimental methods yields an explosion of insights into protein folding mechanisms , 2009, Nature Structural &Molecular Biology.