Probing serpin conformational change using mass spectrometry and related methods

Abstract

The folding, misfolding, and inhibitory mechanisms of serpins are linked to both thermodynamic metastability and conformational flexibility. Characterizing the structural distribution of stability and flexibility in serpins in solution is challenging due to their large size and propensity for aggregation. Structural mass spectrometry techniques offer powerful tools for probing the mechanisms of serpin function and disfunction. In this chapter, we review the principles of the two most commonly employed structural mass spectrometry techniques--hydrogen/deuterium exchange and chemical footprinting--and describe their application to studying serpin flexibility, stability, and conformational change in solution. We also review the application of both hydrogen/deuterium exchange and ion mobility mass spectrometry to probe the mechanism of serpin polymerization and the structure of serpin polymers.

Figure 1

Hydrogens in proteins. The amide hydrogens monitored by H/D exchange are circled.

Figure 2

Overview of the experimental procedure for measuring local H/D exchange rates using mass spectrometry.

Figure 3

Overview of the experimental procedure for monitoring side chain solvent accessibility using radiolytic footprinting.

Figure 4

Distribution of conformational flexibility in the metastable form of α₁-AT determined by HXMS.

Figure 5

Allosteric rigidification resulting from the cavity filling G117F mutation in α₁-AT. F117 is shown as magenta spheres.

Figure 6

A) Example of a double isotopic envelope observed during GuHCl denaturation of α₁-AT. The peptide shown corresponds to residues 64–77. B) Equilibrium unfolding curve for peptide 64–77 constructed from the relative areas under the low (native) and high (denatured) mass peaks in the mass spectra at different GuHCl concentrations.

Figure 7

HXMS derived denaturation curves for peptic fragments derived from different regions of α₁-AT.

Figure 8

Mass spectra for peptides derived from different regions of glycosylated α₁-AT at 0, 1.0, 1.2 and 4 M GuHCl.

Figure 9

Patterns of deuterium uptake in different regions of α₁-AT during the inhibitory conformational change. Red and orange indicate regions that undergo “functional unfolding” during inhibition.

Figure 10

Ion mobility MS of α₁-AT monomer and polymers. After heating, an additional monomeric species with a slightly increased collision cross section (indicating a somewhat expanded structure) is evident.

Figure 11

Changes in deuterium uptake in different regions of α₁-AT upon polymerization.