A Method To Determine Residue-Specific Unfolded-State pK(a) Values from Analysis of Stability Changes in Single Mutant Cycles

作者：Shen Jana K^*

来源：Journal of the American Chemical Society, 2010, 132(21): 7258-+.

DOI：10.1021/ja101761m

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.

• 出版日期2010-6-2