Analyzing Protein Dynamics Using Hydrogen Exchange Mass Spectrometry

Abstract

All cellular processes depend on the functionality of proteins. Although the functionality of a given protein is the direct consequence of its unique amino acid sequence, it is only realized by the folding of the polypeptide chain into a single defined three-dimensional arrangement or more commonly into an ensemble of interconverting conformations. Investigating the connection between protein conformation and its function is therefore essential for a complete understanding of how proteins are able to fulfill their great variety of tasks. One possibility to study conformational changes a protein undergoes while progressing through its functional cycle is hydrogen-1 H/ 2 H-exchange in combination with high-resolution mass spectrometry (HX-MS). HX-MS is a versatile and robust method that adds a new dimension to structural information obtained by e.g. crystallography. It is used to study protein folding and unfolding, binding of small molecule ligands, protein-protein interactions, conformational changes linked to enzyme catalysis, and allostery. In addition, HX-MS is often used when the amount of protein is very limited or crystallization of the protein is not feasible. Here we provide a general protocol for studying protein dynamics with HX-MS and describe as an example how to reveal the interaction interface of two proteins in a complex. Video LinkThe video component of this article can be found at

Hsp70- and Hsp90-Mediated Regulation of the Conformation of p53 DNA Binding Domain and p53 Cancer Variants

“…Local Unfolding Is Induced in p53-DBD at 37 C To explore the kinetics of deuteron incorporation into unstressed human p53-WT-DBD, we incubated p53-DBD for 5, 30, and 60 s in deuterium oxide (D 2 O) buffer at 30 C and analyzed deuteron incorporation as described (Hentze and Mayer, 2013). Most of the analyzed peptic peptides of p53-DBD exchanged from 30%-80% of their amide protons within the first 5 s, underlining the high conformational dynamics of the DBD of p53 (Figure 1A).…”

Mass Spectrometric Approaches to Study Protein Structure and Interactions in Lyophilized Powders

“…The results presented here and elsewhere 27,28,31-33 have demonstrated the ability of these methods to monitor protein conformation with high resolution in the solid environment. Though the critical steps in data analysis do not vary from labeling in solution 34-36 , important considerations during experimental setup and data interpretation are required for solid-state chemical labeling.…”

Mass Spectrometric Approaches to Study Protein Structure and Interactions in Lyophilized Powders

“…The results presented here and elsewhere 27,28,[31][32][33] have demonstrated the ability of these methods to monitor protein conformation with high resolution in the solid environment. Though the critical steps in data analysis do not vary from labeling in solution [34][35][36] , important considerations during experimental setup and data interpretation are required for solid-state chemical labeling.…”