Thermodynamic study of the acid denaturation of barnase and its dependence on ionic strength: evidence for residual electrostatic interactions in the acid/thermally denatured state.
We have investigated the acid denaturation of barnase and its dependence on ionic strength. From the pH dependence of the protein stability, we have obtained information about the titration properties of the native and denatured protein at temperatures ranging from 15 to 60 degrees C in the absence of chemical denaturant. It appears that both the native and the denatured state of barnase titrates at higher pH values in the presence of salt. The observation suggests that charge interactions are present, not only within the native fold but also within the denatured state, and that these interactions contribute to shift the pKa values from those of isolated model compounds. Upon addition of salt these repulsive interactions are shielded, and the electrostatic free energy of the native state, as well as the denatured state, is reduced. Accordingly, we suggest that the thermally denatured state of barnase is not an extended random coil without residue-residue interactions but is sufficiently compact to contain intramolecular charge-charge repulsions. The results further reveal that the native state of barnase contains at least one residue with a highly anomalous pKa value: At pH 0.3, the difference in degree of protonation between the native and the denatured state is still about 1 mol H+/mol protein.
Cambridge Centre for Protein Engineering, England, U.K.
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M
Oliveberg
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Vuilleumier
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Fersht
D001407:Bacillus / Q000201:enzymology; D001426:Bacterial Proteins; D002942:Circular Dichroism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D009994:Osmolar Concentration; D011489:Protein Denaturation; D012260:Ribonucleases / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics
D001426:Bacterial Proteins; D012260:Ribonucleases; C019760:Bacillus amyloliquefaciens ribonuclease