Poster abstracts
Poster number 18 submitted by Pepsi Holmquist
Elucidating the allosteric mechanism of ligand binding to TRAP using native MS
Melody Pepsi Holmquist (Department of Chemistry and Biochemistry, The Ohio State University), Elihu C. Ihms (Department of Chemistry and Biochemistry, The Ohio State University), Paul Gollnick (Department of Biological Sciences, State University of New York at Buffalo), Vicki H. Wysocki (Department of Chemistry and Biochemistry, The Ohio State University), Mark P. Foster (Department of Chemistry and Biochemistry, The Ohio State University)
Abstract:
Cooperativity in ligand binding is crucial for regulation of biological pathways. However, it is challenging to quantify cooperativity due to the multiple identical binding sites and the symmetry of homo-oligomeric proteins. In addition, there is difficulty of measuring accurately and unambiguously the populations of protein-ligand species in order to determine microscopic equilibrium constants. Native MS can quantify homo-oligomeric protein species with different numbers of bound ligands, provided the populations can be directly obtained from the ion counts, and that MS-friendly native buffers do not alter the thermodynamics. By using native mass spectrometry, we measured homotropic binding of tryptophan (Trp) to Bacilllus stearothermophilus TRAP, a homo-oligomeric protein with 11 identical binding sites. To obtain the cooperative microscopic thermodynamics such as coupling free energies and binding affinities of Trp binding to TRAP, we used a nearest-neighbor thermodynamic model. The populations of TRAP species were determined by mass spectra obtained on a high-resolution Orbitrap mass spectrometer under native conditions to quantify the Trp-TRAP states populated at different Trp concentrations. The effect of MS buffers on the structure of TRAP was explored by ion mobility MS experiments, yielding experimental collision cross section (CCS) measurements for various TRAP species. SID-MS was implemented to study the fragments of TRAP with bound Trp. Native MS results were compared to those from solution ITC and NMR experiments in phosphate, AmAc and EDDA buffers. The ITC and NMR solution experiments showed that TRAP retains its native-like structure in MS solutions and that the Trp binding was not inhibited in MS solutions, validating our native MS titration experiments. Using native MS allows us to obtain microscopic thermodynamic constants such as Kd and free energies of the neighboring binding sites, helping us better understand the mechanism of homotropic allostery in ligand binding to TRAP.
Keywords: microscopic thermodynamics, Native MS, ion mobility