Population distributions from native mass spectrometry titrations reveal nearest-neighbor cooperativity in the ring-shaped oligomeric protein TRAP.
2020
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
sites, because the observable [e.g., nuclear magnetic resonance (NMR)
chemical shift changes, fluorescence, and enthalpy] can be altered
by allostery, thereby distorting its proportionality to site occupancy.
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. MS-compatible solutions
did not significantly perturb protein structure or thermodynamics
as assessed by isothermal titration calorimetry and NMR spectroscopy.
Populations of Trpn-TRAP11 states
were quantified as a function of Trp concentration by nMS. The population
distributions could not be explained by a noncooperative binding model
but were described well by a mechanistic nearest-neighbor cooperative
model. Nonlinear least-squares fitting yielded microscopic thermodynamic
constants that define the interactions between neighboring binding
sites. This approach may be applied to quantify thermodynamic cooperativity
in other ring-shaped proteins.
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