Assessing the fractions of tautomeric forms of the imidazole ring of histidine in proteins as a function of pH

<jats:p> A method is proposed to determine the fraction of the tautomeric forms of the imidazole ring of histidine in proteins as a function of pH, provided that the observed <jats:inline-formula> <jats:inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="pnas.1102373108eq1.gif" /> </jats:inline-formula> and <jats:inline-formula> <jats:inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="pnas.1102373108eq2.gif" /> </jats:inline-formula> chemical shifts and the protein structure, or the fraction of H <jats:sup>+</jats:sup> form, are known. This method is based on the use of quantum chemical methods to compute the <jats:sup>13</jats:sup> C NMR shieldings of all the imidazole ring carbons ( <jats:sup>13</jats:sup> C <jats:sup>γ</jats:sup> , <jats:inline-formula> <jats:inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="pnas.1102373108eq3.gif" /> </jats:inline-formula> , and <jats:inline-formula> <jats:inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="pnas.1102373108eq4.gif" /> </jats:inline-formula> ) for each of the two tautomers, N <jats:sup> <jats:italic>δ</jats:italic> 1 </jats:sup> -H and N <jats:sup> <jats:italic>ϵ</jats:italic> 2 </jats:sup> -H, and the protonated form, H <jats:sup>+</jats:sup> , of histidine. This methodology enabled us ( <jats:italic>i</jats:italic> ) to determine the fraction of all the tautomeric forms of histidine for eight proteins for which the <jats:inline-formula> <jats:inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="pnas.1102373108eq5.gif" /> </jats:inline-formula> and <jats:inline-formula> <jats:inline-graphic xmlns:xlink="http://www.w3.org/1999/xlink" xlink:href="pnas.1102373108eq6.gif" /> </jats:inline-formula> chemical shifts had been determined in solution in the pH range of 3.2 to 7.5 and ( <jats:italic>ii</jats:italic> ) to estimate the fraction of tautomeric forms of eight histidine-containing dipeptide crystals for which the chemical shifts had been determined by solid-state <jats:sup>13</jats:sup> C NMR. Our results for proteins indicate that the protonated form is the most populated one, whereas the distribution of the tautomeric forms for the imidazole ring varies significantly among different histidines in the same protein, reflecting the importance of the environment of the histidines in determining the tautomeric forms. In addition, for ∼70% of the neutral histidine-containing dipeptides, the method leads to fairly good agreement between the calculated and the experimental tautomeric form. Coexistence of different tautomeric forms in the same crystal structure may explain the remaining 30% of disagreement. </jats:p>