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Use of 2D HSQC NMR Spectroscopy to Determine Histidine pKa Values in Bacillus subtilis RNase P Protein
- Abstract:
Ionization states of charged residues determine the net charge on a protein and therefore play an important role in protein structure, function, and macromolecular assembly. pKa values are a direct experimental probe of the local environments of ionizable groups. Therefore, knowledge of accurate values can provide information on the strength of electrostatic interactions in a protein and aid in understanding the contribution of ionizable groups to binding and protein stability. Presented here are the pKa values for the three histidine residues of the sulfate-folded state of the protein component of Bacillus subtilis ribonuclease P measured by 1H-13C heteronuclear single quantum correlation NMR spectroscopy. Two of the three histidine residues, His 22 (pKa=6.0) and His 105 (pKa=5.5), titrated with depressed pKa values while the other residue, His 3 (pKa=6.2), had a pKa that was slightly higher than free L-histidine, the model compound. These results indicate that in addition to the overall net charge of the protein, deviations from the model compound pKa value are due to specific local interactions involving the histidine side chains.
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