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

Abstract

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 13 C γ and 13 C δ2 chemical shifts and the protein structure, or the fraction of H þ form, are known. This method is based on the use of quantum chemical methods to compute the 13 C NMR shieldings of all the imidazole ring carbons ( 13 C γ , 13 C δ2 , and 13 C ϵ1 ) for each of the two tautomers, N δ1 -H and N ϵ2 -H, and the protonated form, H þ , of histidine. This methodology enabled us (i) to determine the fraction of all the tautomeric forms of histidine for eight proteins for which the 13 C γ and 13 C δ2 chemical shifts had been determined in solution in the pH range of 3.2 to 7.5 and (ii) to estimate the fraction of tautomeric forms of eight histidine-containing dipeptide crystals for which the chemical shifts had been determined by solid-state 13 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.histidine protonation | histidine tautomers | pH effect | side-chain conformation A mong all 20 naturally occurring amino acids, histidine (His) is a unique residue for a number of reasons, among others because ∼50% of all enzymes use His in their active sites (1). This is mainly because of the chemical versatility of its imidazole ring, which includes two neutral, chemically distinct forms, and a protonated form, referred to as N δ1 -H and N ϵ2 -H tautomers, and H þ , respectively, with one form favored over the other by the protein environment and pH. Moreover, His with a pK°of 6.6 (2) titrates around neutral pH, allowing the deprotonated nitrogen of its imidazole ring to serve as an effective ligand for metal binding (3). In particular, it has been suggested that tautomerization and variations of χ1 of His are crucial parts of the proton-transfer process (4). In addition, it has also been recognized (5) that many imidazole-containing ligands could exhibit large chemical-shift variations when bound to a molecular target, such as a protein, offering valuable information about changes in the local structure of the ligand or target. Hence, characterization of the tautomers of drug molecules could have important consequence in the pharmaceutical industry. It is particularly interesting that the most abundant type of tautomers in the Cambridge Structural Database (CSD) correspond to derivatives of azoles, such as pyrazoles, imidazoles, etc. (6).Since chemical shifts were first observed by Ar...

The Influenza M2 Cytoplasmic Tail Changes the Proton-Exchange Equilibria and the Backbone Conformation of the Transmembrane Histidine Residue to Facilitate Proton Conduction

“…The Cγ chemical shift is also resolved among the three states, adopting the most downfield values (136–138 ppm) for the τ tautomer, the most upfield values (126–129 ppm) for the π tautomer, and intermediate values (129– 132 ppm) for cationic histidine. Using the Cγ and Cδ2 chemical shift difference as a criterion, the τ and π tautomers are distinguished by large (> 20 ppm) and small (< 5 ppm) chemical shift differences, respectively, while cationic histidine has intermediate chemical shift difference of 10–15 ppm 41 .…”

Measurement of histidine pKavalues and tautomer populations in invisible protein states

“…Further, these two shifts are highly anticorrelated, suggesting that their difference could potentially remove local effects arising from interactions with neighboring residues, and therefore provide an excellent probe of the tautomeric state of the imidazole ring. Although a number of studies, both experimental and computational, have provided estimates of 13 C shifts in each of the δ, «, and + states (7,52,56,57,63), the majority have been performed on small peptides or amino acids, and we were interested in obtaining consensus values that would be appropriate in the context of a protein environment. Fig.…”

Synthesis, spectroscopic and computational characterization of the tautomerism of pyrazoline derivatives from chalcones

“…In this context, NMR is a widely used and very powerful tool for the characterization of tautomeric equilibrium for some important compounds such as histidine and D-fructose, among others [20,21]. The use of NMR techniques for the description of tautomeric equilibrium relies on chemical systems that present slow enough proton exchange for both systems to be measured in solution [22,23].…”