Poster abstracts
Poster number 21 submitted by Pepsi Holmquist
Probing the mechanism of homotropic allostery in TRAP using native mass spectrometry
Melody Pepsi Holmquist (Ohio State Biochemistry Program, 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 Wysocki (Department of Chemistry and Biochemistry, The Ohio State University), Mark P. Foster (Department of Chemistry and Biochemistry, The Ohio State University)
Abstract:
Homotropic allostery describes the cooperative binding of ligands to a macromolecule with multiple binding sites for the same ligand. In the case of positive cooperativity, binding of one ligand increases the affinity of the macromolecule for additional ligands; oxygen binding by hemoglobin is a classic example. In order to understand the mechanism of such allostery, one must first quantify the thermodynamic coupling between ligand binding sites. This is generally difficult because experimental measurements of affinity yield an apparent macroscopic parameter that is composed of many microscopic parameters, including the site-site coupling terms of interest. We are developing and testing an approach to provide access to those microscopic interaction terms by studying allostery in trp RNA binding attenuation protein (TRAP). TRAP is an oligomeric protein with 11 identical binding sites for the ligand tryptophan, Trp. TRAP becomes activated upon Trp binding and binds to the 5’ leader region of the trp mRNA, resulting in both transcription attenuation and translation inhibition, presenting a regulated feedback loop that controls tryptophan biosynthesis in Bacillus cells. A key difficulty with accurately obtaining microscopic parameters describing cooperativity is that in typical binding measurements, where binding isotherms are constructed from ligand-dependent changes in fluorescence, NMR chemical shift, or enthalpy, allostery may distort the proportion between the measurable and the number of the bound ligands. However, the mass of a protein with n ligands bound is not biased by allosteric effects and reports directly the number of bound ligands. As long as populations can be accurately obtained from the ions counted by MS detectors, MS can be used to measure populations of bound species, thereby increasing the accuracy with which microscopic parameters can be obtained. By using native and ion mobility mass spectrometry, we measured concentration dependent binding of Trp to TRAP and use a nearest-neighbor thermodynamic model to quantify the microscopic thermodynamics of the cooperativity in TRAP. In addition, we used solution measurements (ITC) in MS-compatible buffers to test and validate native MS as a tool to measure microscopic thermodynamic parameters.
Keywords: microscopic thermodynamics, native mass spectrometry, isothermal titration calorimetry