We have determined the pKA values of the 12 carboxyl residues in the native and denatured state of barnase by a combination of thermodynamic measurements on mutants of charged residues and NMR titration data. The pKA values of the 11 residues titrating under folding conditions (above pH 2.2) were determined by two-dimensional 1H NMR. The pKA value of the remaining residue, Asp 93 which forms a salt link with Arg 69 and titrates at much lower pH values, was determined by changes in the pH dependence of the stability of the protein upon mutation to Asn: pKAsp93A at low ionic strength (50 mM) and pKAsp93A at high ionic strength (600 mM). The overall titration of the native state is nonideal, and the protein retains fractionally ionized residues other than Asp 93 throughout the experimental pH range of 0.2-6.3. Protonation events taking place at pH values below 2 were further characterized by the pH dependence of the unfolding kinetics of wild-type and charge-mutant proteins. By comparing the observed pH dependence of the protein stability with that calculated from the pKA values for the native protein, we demonstrate that the pKA values of the denatured state are significantly lower than those reported for model compounds: the pKA values of the denatured state appear on average 0.4 units lower than previous estimates in the presence of chemical denaturant. The results have direct implications for calculations of the energetics of proton equilibria and suggest that the acid/thermally denatured state is not an extended coil where the residues are isolated from one another by the intervening solvent but is compact and involves intramolecular charge repulsion. Study holds ProTherm entries: 5069, 5070, 5071, 5072, 5073 Extra Details: charged residues; protonation; unfolding kinetics; solvent;,intramolecular charge repulsion
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:28 p.m.
|Number of data points||9|
|Proteins||Ribonuclease ; Ribonuclease|
|Assays/Quantities/Protocols||Experimental Assay: dG ; Derived Quantity: ddG|
|Libraries||Mutations for sequence AQVINTFDGVADYLQTYHKLPDNYITKSEAQALGWVASKGNLADVAPGKSIGGDIFSNREGKLPGKSGRTWREADINYTSGFRNSDRILYSSDWLIYKTTDHYQTFTKIR|