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.
Shen
Jana K
JK
Department of Chemistry and Biochemistry, University of Oklahoma, Norman, Oklahoma 92037, USA. jana.k.shen@ou.edu
D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics; D056004:Molecular Dynamics Simulation; D009154:Mutation; D017510:Protein Folding; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics
D000596:Amino Acids; D012269:Ribosomal Proteins; C049650:ribosomal protein L9