Influence of coulombic repulsion on the dissociation pathways and energetics of multiprotein complexes in the gas phase

Abstract

Thermal dissociation experiments, implemented with blackbody infrared radiative dissociation and Fourier-transform ion cyclotron resonance mass spectrometry, are performed on gaseous protonated and deprotonated ions of the homopentameric B subunits of Shiga toxin 1 (Stx1 B 5 ) and Shiga toxin 2 (Stx2 B 5 ) and the homotetramer streptavidin (S 4 ). Dissociation of the gaseous, multisubunit complexes proceeds predominantly by the loss of a single subunit. Notably, the fractional partitioning of charge between the product ions, i.e., the leaving subunit and the resulting multimer, for a given complex is, within error, constant over the range of charge states investigated. The Arrhenius activation parameters (E a , A) measured for the loss of subunit decrease with increasing charge state of the complex. However, the parameters for the protonated and deprotonated ions, with the same number of charges, are indistinguishable. The influence of the complex charge state on the dissociation pathways and the magnitude of the dissociation E a are modeled theoretically with the discrete charge droplet model (DCDM) and the protein structure model (PSM), wherein the structure of the subunits is considered. Importantly, the major subunit charge states observed experimentally for the Stx1 B 5 nϮ ions correspond to the minimum energy charge distribution predicted by DCDM and PSM assuming a late dissociative transition-state (TS); while for structurally-related Stx2 B 5 nϩ ions, the experimental charge distribution corresponds to an early TS. It is proposed that the lateness of the TS is related, in part, to the degree of unfolding of the leaving subunit, with Stx1 B being more unfolded than Stx2 B. PSM, incorporating significant subunit unfolding is necessary to account for the product ions observed for the S 4 nϩ ions. The contribution of Coulombic repulsion to the dissociation E a is quantified and the intrinsic activation energy is estimated for the first time. T he majority of cellular proteins exist and function as multimeric complexes. Consequently, the characterization of protein assemblies, their structure, stability, and biological function represent important analytical objectives. Mass spectrometry (MS), with its speed, sensitivity, and specificity, combined with electrospray ionization (ES), is an established tool for detecting specific multiprotein complexes in solution [1], the real time monitoring their assembly/disassembly [2] and subunit exchange reactions [3,4], and identifying and quantifying their interactions with other biopolymers, as well as other ligands and cofactors [5]. Several comprehensive reviews of the applications of ES/MS to protein complexes have appeared, [1,[5][6][7][8][9], including a recent review by Robinson and coworkers [8].Combined with gas-phase ion activation techniques, which can be used to break the noncovalent interactions and, thereby, release individual subunits from the multiprotein complexes, ES/MS also holds promise as a tool for determining the subunit composition and, perhaps, ...

Theoretical investigations of the dissociation of charged protein complexes in the gas phase

“…This indicates that one streptavidin monomer is sufficiently flexible so as to unfold to produce a surface area commensurate with the remaining trimer fragment ion. This interpretation is consistent with the electrostatic model calculations of Sinelnikov et al [15], who showed that a partially unfolded monomer subunit could account for the observed charge ratios. Interpreted in another way, the monomer need unfold only to the extent of making its surface area commensurate with the remaining trimer fragment ion.…”

“…As the charge asymmetry increases, so does the barrier to dissociation. This is also consistent with the experimental results of Sinelnikov et al [15] who assert that the long-range repulsion between charged groups dominate the dissociation of a number of protein complexes, and who sought to understand the relationship between measured activation energies and some calculated values of this repulsion. It should also be noted that this long-range repulsion is generally the same regardless of how the charges q 1 and q 2 are distributed, since the repulsion between two uniformly charged spheres with charges q 1 and q 2 is the same as that between two point charges.…”

“…For example, tetrameric complexes of protonated streptavidin ions are found to fragment in a symmetric fashion, [10,15] with S 4 ϩn ions producing predominantly S 3 ϩn⁄2 n n and S ϩn⁄2 n n fragments, with n varying between 14 and 18. Fluctuations around these limits of one unit of charge are also observed.…”

“…One challenge in using mass spectrometry for the general analysis of protein complexes is understanding the dissociation mechanism of these complexes in the gas phase. Many groups [5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] have reported asymmetric dissociation behaviour for large multimeric proteins. A small subunit, typically a protein monomer, is ejected from the complex during dissociation, with the monomer carrying away a disproportionate amount of charge for its relative mass.…”

“…Klassen and coworkers [13][14][15][16] have reported a series of studies, mostly on complexes of the Shiga toxin and streptavidin complexes, in which careful thermodynamic measurements have been made employing the blackbody infrared radiative dissociation (BIRD) technique. This has been coupled with several model calculations, also based upon electrostatic calculations of charges distributed both on idealized spheres, and on protein structures.…”