Bridging protein structure, dynamics, and function using hydrogen/deuterium-exchange mass spectrometry

Abstract

Much of our understanding of protein structure and mechanistic function has been derived from static high-resolution structures. As structural biology has continued to evolve it has become clear that high-resolution structures alone are unable to fully capture the mechanistic basis for protein structure and function in solution. Recently Hydrogen/Deuterium-exchange Mass Spectrometry (HDX-MS) has developed into a powerful and versatile tool for structural biologists that provides novel insights into protein structure and function. HDX-MS enables direct monitoring of a protein's structural fluctuations and conformational changes under native conditions in solution even as it is carrying out its functions. In this review, we focus on the use of HDX-MS to monitor these dynamic changes in proteins. We examine how HDX-MS has been applied to study protein structure and function in systems ranging from large, complex assemblies to intrinsically disordered proteins, and we discuss its use in probing conformational changes during protein folding and catalytic function. STATEMENT FOR A BROAD AUDIENCE: The biophysical and structural characterization of proteins provides novel insight into their functionalities. Protein motions, ranging from small scale local fluctuations to larger concerted structural rearrangements, often determine protein function. Hydrogen/Deuterium-exchange Mass Spectrometry (HDX-MS) has proven a powerful biophysical tool capable of probing changes in protein structure and dynamic protein motions that are often invisible to most other techniques.

Keywords: conformational switching; conformational transitions; hydrogen/deuterium-exchange mass spectrometry (HDX-MS); intrinsic disorder; structural dynamics; structural rearrangements.

Figure 1

Protein structural dynamics and motion monitored by HDX‐MS. Continuous labeling HDX‐MS probes the accessibility of backbone amide hydrogens (blue circles) by their exchange with deuterium in solution. Under equilibrium conditions the majority of protein structural dynamics and motion manifests as “EX2 kinetics” (a) where deuterium is gradually incorporated across the protein backbone in a manner directly related to the local structural dynamics and changes in amide accessibility. Examples of peptide specific HDX‐MS data with representative mass spectra show the gradual incorporation of deuterium over time. Peptide segments in highly structured regions with strong hydrogen bonding networks (green beta sheet) take up deuterium much slower than regions with exposed and accessible amides (pink loop) and those undergoing dynamic motions (blue helix) that occur faster than the labeling rate. (b) When these dynamic structural changes are slower than the labeling rate (e.g., a reversible interconversion or conformational change) they produce unique and resolvable HDX states that manifest as “EX1 kinetics.” Here the protein reversibly interconverts between conformations with a protected (blue) or exposed (orange) helical domain. Analysis of the mass spectra reveals the equilibrium distribution of these two states and their respective half‐lives

Figure 2

Elucidating the mechanism of protein folding by pulse labeling HDX‐MS. The folding of maltose binding protein (MBP) was monitored by pulse labeling HDX‐MS where unfolded and fully deuterated MBP was diluted to initiate folding. Using a quench flow system the samples were rapidly labeled during folding with a ~millisecond pulse of H2O (D–H exchange) resulting in bimodal isotopic distributions showing the formation of secondary structure, and resistance to exchange, as each peptide folds (e–g). Analysis of all peptides folding kinetics revealed the formation of an obligate “fast folding” intermediate state (blue traces and highlights a–e). Subsequent folding events radiated outward from the structural core formed by the fast folding intermediate and only a single distinct folding pathway was observed (a and b). The contact map in (b) highlights the long‐range contacts formed by the fast folding intermediate and how each subsequent folding event was seeded by the formation of this structural core. Peptide specific folding kinetics mapped onto the crystal structure for the native folded state (c and d) further highlight how peptides nearest to the fast folding intermediate and structural core (c, d, and e blue highlights) fold sequentially radiating outward from the core (c, d, f, and g). Source: Adapted from Reference 32 with permission

Figure 3

Ligand induced disorder to order transition is critical for catalytic function in CyaA. The structure of free CyaA (AC) eluded previous structural characterization as large sections of the protein are intrinsically disordered. By HDX‐MS it was observed that a 75 amino acid long region, previously seen as helical in the ligand bound structure, was intrinsically disordered in the apo state and becomes ordered upon binding with CaM (a and b). Purple indicates helices with dramatic increases in protection from deuteration, and green indicates regions where a dynamic event occurred upon ligand binding. Differences in percent deuteration on AC and CaM upon binding CaM and AC, respectively, mapped onto the crystal structure (c and d). These data highlight how CaM induces formation of structure in AC upon binding, however that structure is highly dynamic across the H helicies. In contrast the catalytic site remains largely unperturbed as it is primed for high catalytic turnover. Source: Adapted from Reference 52 with permission

Figure 4

HDX‐MS reveals a novel functional state in protein kinase A ternary complex. Hydrolysis of cAMP by the protein kinase A (PKA) and phosphodiesterase (PDE) ternary complex was monitored in real time by HDX‐MS. A highly broad mass envelope appeared over time in the ternary complex (top right spectra). Analysis of each component and their possible assemblies (spectra shown on the right) could not explain the distribution seen in the ternary complex indicating this is a unique and catalytically active structural state. Source: Adapted from Reference 54 with permission

Figure 5

Dynamic conformational changes in the integral membrane protein Pgp. The ABCB1 transporter P‐glycoprotein P (Pgp) reversibly interconverts between inward and outward facing conformational states to facilitate transport of ligand across the cell membrane. HDX‐MS of Pgp in lipid nanodics and detergent micelles revealed conformational transitions occurring on multiple timescales across Pgp indicating the presence of more than two discrete conformational states (a–c). Binomial fitting of bimodal isotopic distributions enabled the deconvolution of multiple coexisting HDX populations and analysis of their conformational kinetics (a). In nanodiscs peptides throughout Pgp exhibited EX1 kinetics on three distinct timescales; fast (red), moderate (yellow), and slow (purple). In contrast Pgp reconstituted in detergent micelles contained fewer peptides exhibiting EX1 kinetics and did not contain those peptides where fast transitions were observed in nanodiscs (a and b). Peptides exhibiting EX1 kinetics mapped onto Pgp structure by their relative timescales (c). Source: Adapted from Reference 59 with permission