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

• histidine
• histidine protonation
• acid dissociation constant
• mass spectrometry
• protein structural biology