Probing allosteric mechanisms using native mass spectrometry

Abstract

Native mass spectrometry (MS) and ion mobility MS provide a way to discriminate between various allosteric mechanisms that cannot be distinguished using ensemble measurements of ligand binding in bulk protein solutions. Native MS, which yields mass measurements of intact assemblies, can be used to determine the values of ligand binding constants of multimeric allosteric proteins, thereby providing a way to distinguish, for example, between concerted and sequential allosteric models. Native MS can also be employed to study cooperativity owing to ligand-modulated protein oligomerization. The rotationally averaged cross-section areas of complexes obtained by ion mobility MS can be used to distinguish between induced fit and conformational selection. Native MS and its allied techniques are, therefore, becoming increasingly powerful tools for dissecting allosteric mechanisms.

Figure 1. MS techniques for studying allosteric mechanisms.

Schematic representation of the native MS (left) and IM (right) approaches. Native MS is based on the ability to transfer intact protein complexes to the gas phase, while maintaining weak non-covalent interactions between protein subunits and bound ligands. Insight into allosteric mechanisms can be gained by determining the protein populations with different numbers of bound ligand molecules (bottom left panel). IM measures the time it takes for an ion to travel through a tube filled with an inert gas. The ion’s transit time depends not only on mass and charge, but also on its overall shape: an assembly with a large volume will experience more collisions with the gas and, therefore, travel more slowly than a complex with the same mass but a more compact structure. The measured drift times (dt) can be converted into collision cross-sections which, in turn, can be related to the conformation of the analyzed assembly. IM can be used to characterize structural changes in the conformational ensemble upon ligand binding.

Figure 2. Electrospray ionization MS can reveal the number of ATP molecules bound to GroEL.

(A) Superposition of spectra acquired on a modified high mass QToF instrument in the presence of different concentrations of ATP (the region of the 57⁺ charge state is displayed). The peaks are labeled according to the number of bound ATP molecules to which they correspond. The increase in the number of bound ATP molecules as a function of ATP concentration reflects the step-wise manner of ATP binding to GroEL (reproduced with permission from [18]). (B) Mass spectra acquired on an extended mass range Exactive Plus Oribtrap showing the 70⁺ charge state of GroEL unbound (black) or incubated with ADP (red) or ATP (blue). The high mass resolution allows the number of bound nucleotides (ATP or ADP + Na+, Δm/z 6.4 or 7.6 Th) to be counted, as indicated (reproduced with permission from [17]).

Figure 3. Recounting protein populations according to specific binding.

The illustration shows for a dimer with two specific and three non-specific ligand binding sites how the populations with different numbers of bound ligand molecules are counted before (A) and after (B) taking into account non-specific binding. The protein populations with 0, 1 and 2 ligand molecules bound at specific sites are shown in red, blue and green respectively. Contributions from both specific and non-specific binding are included in each population with a given number of bound ligand molecules before the correction for non-specific binding is implemented. Following the correction, all the protein molecules with a given number of specifically bound ligand molecules are counted together regardless of the number of non-specifically bound ligand molecules. Listed below each pole of the abacus are the relative weights of the different populations that it counts.

Figure 4. Scheme describing the concerted (MWC) and sequential (KNF) allosteric models for a tetramer.

According to the concerted model, the oligomer in its apo state is in equilibrium between low (T) and high (R) affinity states for the ligand (L = [R]/[T]). In the case of the concerted model with exclusive binding of the ligand to the R state (A), the values of the intrinsic binding constants corresponding to all the successive binding steps will be the same except that of the first site which will differ by a factor of L/(1+L). In the case of the concerted model with nonexclusive binding of the ligand to both the T and R states (B), the values of the intrinsic binding constants corresponding to the successive binding steps will all differ from each other but they will form a series that can be expressed as a function of L and the respective ligand binding constants of the T and R states, K_T and K_R. In the case of the sequential model (C), a simple relationship between the values of the N binding constants is not necessarily expected.