Population Distributions from Native Mass Spectrometry Titrations Reveal Nearest-Neighbor Cooperativity in the Ring-Shaped Oligomeric Protein TRAP

Abstract

Allostery pervades macromolecular function and drives cooperative binding of ligands to macromolecules. To decipher the mechanisms of cooperative ligand binding it is necessary to define, at a microscopic level, the thermodynamic consequences of binding of each ligand to its energetically coupled site(s). However, extracting these microscopic constants is difficult for macromolecules with more than two binding constants. This goal is complicated because the observable (e.g., NMR chemical shift changes, fluorescence, enthalpy) can be altered by allostery, thereby distorting its proportionality to populations of states. Because it measures mass, native mass spectrometry (MS) can directly quantify the populations of homo-oligomeric protein species with different numbers of bound ligands, provided the populations are proportional to ion counts and that MS-compatible electrolytes do not alter the overall thermodynamics. These measurements can help decipher allosteric mechanisms by providing unparalleled access to the statistical thermodynamic partition function. We used native MS (nMS) to study the cooperative binding of tryptophan (Trp) to Bacillus stearothermophilus trp RNA-binding attenuation protein (TRAP), a ring-shaped homo-oligomeric protein complex with 11 identical binding sites. Mass spectrometrycompatible solutions did not significantly perturb protein structure and thermodynamics as assessed by ITC and NMR spectroscopy. Populations of Trpn-TRAP11 states were quantified as a function of Trp concentration by native mass spectrometry.Population distributions cannot be explained by a non-cooperative binding model but are well described by a nearest neighbor cooperative model. Non-linear least-squares fitting of the populations to a mechanistic model yielded microscopic thermodynamic constants that define the interactions between neighboring binding sites that result in homotropic cooperativity in Trp binding to TRAP. File list (2) download file view on ChemRxiv TrpTRAP_manuscript_mainbody-mf.pdf (1.22 MiB) download file view on ChemRxiv PLH_20200428_TrpTRAP_suppinfo.pdf (0.96 MiB)

Thermodynamic coupling between neighboring binding sites in homo-oligomeric ligand sensing proteins from mass resolved ligand dependent population distributions

“…Such a pattern is consistent with strong positive cooperativity (i.e., Figure 2B). Population distributions for Bst TRAP were similar to those previously described 15 , with a high population of intermediate states consistent with weak cooperativity. The distributions for Bst TRAP Δ71 were intermediate between the other two, reflecting intermediate cooperativity (Figure 5, middle).…”

“…Before thermodynamic analysis of the MS titration data, protein concentrations were corrected by fitting summed bound fraction at each Trp concentration obtained from integration of peak intensities, with an independent sites model: ‫ܯ‬ ‫ܮ‬ ՜ ‫,ܮܯ‬ where M is a TRAP protomer (binding site) and L is the Trp ligand: ܻ ൌ ାெ ା ିඥሺ ାெ ା ሻ మ ିସெ ଶெ ; total ligand L t was assumed to be accurate, while total site concentration M t and apparent dissociation constant K d were fit parameters. 15 This fit yielded the binding site concentration M t that was in general about 33% higher than initial estimated concentration (Table S3, Figure S8).…”

“…The gene of Bst TRAP WT (WP_033013997.1) and the C-terminal-five-residue-truncated Bst TRAP ∆71 were cloned on pET17b and pET9a (Novagen, Inc.), respectively, as previously described. 15,23 Both proteins shared the same preparation protocol. Proteins were expressed in E. coli BL21(DE3) cells, grown at 37˚C in LB, and induced at OD 600 = 0.4 to 0.6 with 1 mM IPTG.…”

Surface-induced dissociation of protein complexes on a cyclic ion mobility spectrometer

“…1,2 nMS is a powerful technique that allows the study of proteins and protein complexes in the gas phase by kinetic trapping of solution structures during the electrospray process, 3 enabling the determination of stoichiometry, ligand binding, oligomeric state, and, when coupled with ion mobility, collision cross section measurements (which can be compared to structures determined by other structural biology tools or computational structure predictions). [4][5][6] Coupling native mass spectrometry with activation methods such as collision-induced dissociation (CID), electron capture dissociation (ECD), electron transfer dissociation (ETD), and ultraviolet photodissociation (UVPD) allows structure and sequence information to be determined. [7][8][9][10] Surface-induced dissociation (SID) allows ligand binding and subunit connectivity information to be obtained as well.…”

Surface-Induced Dissociation of Anionic vs Cationic Native-like Protein Complexes

“…TRAP was expressed in Escherichia coli BL21(DE3) cells, purified and refolded as previously described. 32,33 Samples were buffer exchanged into 200 mM AmAc or 400 mM AmAc, respectively using Biorad micro P6 spin columns. Human proteasome was purified by FLAG affinity purification from HEK293 cells as previously described and free FLAG peptide was present in the analyzed samples.…”