Significance of Histidine Hydrogen-Deuterium Exchange Mass Spectrometry in Protein Structural Biology

Abstract

Histidine residues play crucial roles in shaping the function and structure of proteins due to their unique ability to act as both acids and bases. In other words, they can serve as proton donors and acceptors at physiological pH. This exceptional property is attributed to the side-chain imidazole ring of histidine residues. Consequently, determining the acid-base dissociation constant (Ka) of histidine imidazole rings in proteins often yields valuable insights into protein functions. Significant efforts have been dedicated to measuring the pKa values of histidine residues in various proteins, with nuclear magnetic resonance (NMR) spectroscopy being the most commonly used technique. However, NMR-based methods encounter challenges in assigning signals to individual imidazole rings and require a substantial amount of proteins. To address these issues associated with NMR-based approaches, a mass-spectrometry-based method known as histidine hydrogen-deuterium exchange mass spectrometry (His-HDX-MS) has been developed. This technique not only determines the pKa values of histidine imidazole groups but also quantifies their solvent accessibility. His-HDX-MS has proven effective across diverse proteins, showcasing its utility. This review aims to clarify the fundamental principles of His-HDX-MS, detail the experimental workflow, explain data analysis procedures and provide guidance for interpreting the obtained results.

Keywords: acid dissociation constant; histidine; histidine protonation; mass spectrometry; protein structural biology.

Figure 1

Atom nomenclature of histidine residue. Greek letters (in red) are typically employed to denote the atomic positions of a histidine residue. Alternatively, a numbering system is often used to specify the atomic positions of the histidine imidazole ring, as shown in blue.

Scheme 1

Acid-base equilibrium between the imidazole group ($\mathrm{I}\mathrm{m}$) and the imidazolium group ($\mathrm{I}\mathrm{m}·{\mathrm{H}}^{+}$) with the dissociation constant $K_a$. The equilibrium arrows (⇌) signify that the two species on each side are in equilibrium, while the resonance arrow (↔) suggests that the two species on either side are resonance structures of each other.

Figure 2

The idealized NMR titration curve (red line) of His C-2 proton resonance. The variations in chemical shifts, denoted by the distances between blue lines (${\delta }_{Im·H+}$ and ${\delta }_{obs}$, and ${\delta }_{obs}$ and ${\delta }_{Im}$), scale proportionally with the concentrations [$Im$] and $\left[Im·H^{+}\right]$, respectively. The midpoint of the titration curve is represented by $\left({\delta }_{Im·H+}+{\delta }_{Im}\right)/2$.

Scheme 2

The mechanism of HDX reaction at the C-2 position of the imidazole group. The rate-determining step corresponds to the base-catalyzed abstraction of the C-2 proton of the imidazolium group ($\mathrm{I}\mathrm{m}·{\mathrm{D}}^{+}$) to form an ylide intermediate. In this scheme, the possible resonance hybrid of the ylide—a carbene—is omitted for simplicity. Protons (H) and deuterons (D) are shown in blue and red letters, respectively.

Figure 3

The pH titration curve of His12 of RNase A and its theoretical curve calculated using Equation (6).

Figure 4

The change in the cluster of isotopic peaks before (red) and after (blue) HDX reaction.

Figure 5

$\mathrm{p}{K}_{\mathrm{a}}$ dependence of the intrinsic maximum HDX rates, ${}^{i}k_{\phi }^{\mathrm{m}\mathrm{a}\mathrm{x}}$. pH dependence of the HDX rate, $k_{\phi }$, for the imidazole groups in histamine (red), Ac-His-NHMe (blue), Ac-His-OH (green) and IPA (1H-imidazole-5-propanoic acid, black) is shown. Experimentally obtained $\mathrm{p}{K}_{\mathrm{a}}$ values and intrinsic maximum HDX rates, ${}^{i}k_{\phi }^{max}$, for the four imidazole derivatives are shown. This figure was modified from Figure 2 in Ref. [28].

Figure 6

General workflow of His-HDX-MS experiments.

Figure 7

Sketch of a double sigmoidal titration curve expected to be obtained from the HDX experiment on a His residue influenced by the ionization state of the amino group of a Lys residue in neutral (Lys) and protonated (Lys+) forms. The dissociation constants pK₁ and pK₄ reflect the dissociations of the imidazolium group when Lys is protonated and deprotonated, respectively. Under these conditions, the rate constants $\left(k_{\phi }^{\mathrm{m}\mathrm{a}\mathrm{x}}\right)$₁ and $\left(k_{\phi }^{\mathrm{m}\mathrm{a}\mathrm{x}}\right)$₄ correspond to the $k_{\phi }^{\mathrm{m}\mathrm{a}\mathrm{x}}$ values of the HDX reaction occurring for protonated and neutral forms of Lys, respectively. Hence, the total HDX rate constant, $\left(k_{\phi }^{\mathrm{m}\mathrm{a}\mathrm{x}}\right)$₁₊₄ is the sum of $\left(k_{\phi }^{\mathrm{m}\mathrm{a}\mathrm{x}}\right)$₁ and $\left(k_{\phi }^{\mathrm{m}\mathrm{a}\mathrm{x}}\right)$₄.

Scheme 3

The microscopic dissociation equilibria of systems consisting of His and Lys in their protonated and non-protonated forms. In System I, both the imidazole group of His and the ε-amino group of Lys are protonated, designated as His+ and Lys+. In System II, the imidazole group of His is deprotonated, while the ε-amino group of Lys is protonated (His and Lys+). In System III, the imidazole group of His is protonated, while the ε-amino group of Lys is deprotonated (His+ and Lys). In System IV, both the imidazole group of His and the ε-amino group of Lys are deprotonated (His and Lys). The arrows indicate the reversibility among the systems. It is assumed that these two residues are not necessarily exchanging proton directly but are sufficiently close to interact electrostatically. The His+ form in Systems I and III serves as the sole species, which can react with the ${\mathrm{O}\mathrm{D}}^{-}$ ion to proceed to the exchange of the C-2 proton (see Scheme 2).

Figure 8

PF versus ASA plot for five His residues of E. coli DHFR. Data points plotted in blue denote His residues in apo-DHFR (a), and those plotted in red denote His residues in complex with folate and NADP⁺ (c). This plot was produced using data contained in Table 2 in Ref. [28]. The linear regression line was established by excluding the data points corresponding to His149(a) and His114(c), leading to an R-squared value of 0.79.

Scheme 4

A possible mechanism of the extremely slow C-2 HDX reaction in His48. The network of hydrogen bonds connecting Thr82, His48 and Asp14 virtually restricts the imidazole group of His48 to the electrically neutral state due to the overwhelmingly lower acidity of the hydroxy group (pK_a > 14) than the imidazolium group. The C-2 proton (blue) is replaced by deuteron (red) in this reaction (see also Scheme 2). Note that HDX reaction can occur only through the imidazolium cation, which can populate only marginally in this situation. PDB code 1RPH was used to create this scheme.