Determinants of Gas-Phase Disassembly Behavior in Homodimeric Protein Complexes with Related Yet Divergent Structures

Abstract

The overall structure of a protein-protein complex reflects an intricate arrangement of non-covalent interactions. While intramolecular interactions confer secondary and tertiary structure to individual subunits, intermolecular interactions lead to quaternary structure - the ordered aggregation of separate polypeptide chains into multi-subunit assemblies. The specific ensemble of non-covalent contacts dictates the stability of subunit folds, enforces protein-protein binding specificity, and determines multimer stability. Consequently, non-covalent architecture is likely to play a role in the gas-phase dissociation of these assemblies during tandem mass spectrometry (MS/MS). To further advance the applicability of MS/MS to analytical problems in structural biology, a better understanding of the interplay between the structures and fragmentation behaviors of non-covalent protein complexes is essential. The present work constitutes a systematic study of model protein homodimers (bacteriophage N15 Cro; bacteriophage λ Cro; bacteriophage P22 Arc) with related but divergent structures, both in terms of subunit folds and protein-protein interfaces. Because each of these dimers has a well-characterized structure (solution and / or crystal structure), specific non-covalent features could be correlated with gas-phase disassembly patterns as studied by collision-induced dissociation, surface-induced dissociation, and ion mobility. Of the several respects in which the dimers differed in structure, the presence or absence of intermolecular electrostatic contacts exerted the most significant influence on the gas-phase dissociation behavior. This is attributed to the well-known enhancement of ionic interactions in the absence of bulk solvent. Because salt bridges are general contributors to both intermolecular and intramolecular stability in protein complexes, these observations are broadly applicable to aid in the interpretation or prediction of dissociation spectra for non-covalent protein assemblies.

An Electrostatic Charge Partitioning Model for the Dissociation of Protein Complexes in the Gas Phase

“…(2) Intramolecular crosslinks diminish the extent of charge asymmetry, in line with the expectation that subunit unfolding will be impeded under these conditions [33,37]. (3) Because unfolding is a time-dependent process, symmetric partitioning is favored when employing surfaceinduced dissociation (a picosecond process [49,50]) instead of CID, which proceeds on a microsecond time scale [30]. (4) Ion mobility measurements on collisionally activated multi-protein complexes confirm the presence of semiunfolded intermediate structures [35,51,52].…”

Electron‐capture dissociation and ion mobility mass spectrometry for characterization of the hemoglobin protein assembly

“…The adult human hemoglobin assembly contains two pairs of polypeptide chains, α and β, and four heme groups containing iron ions. The assemblies have been investigated by MS‐based approaches for some time . Native spray of the intact assemblies generated charge states spanning 14+ to 18+ with 16+ the most abundant on both the QToF and the FTICR instruments that we used in this research (Fig.…”

The Role of Salt Bridges, Charge Density, and Subunit Flexibility in Determining Disassembly Routes of Protein Complexes

“…References: 1 (van den Heuvel et al., 2006); 2 (Boeri Erba et al., 2010); 3 (Zhou et al., 2013); 4 (Kükrer et al., 2012); 5 (Dodds et al., 2011); 6 (Loo, 2001); 7 (Versluis et al., 2001); 8 (Aquilina, 2009); and 9 (Fitzgerald et al., 1996). …”