Poster abstracts
Poster number 13 submitted by Pepsi Holmquist
Native Mass Spectrometry Reveals Population Shifts from Allosteric Coupling Between Small Molecule and RNA Binding to the Gene Regulatory Protein TRAP
Melody Pepsi Holmquist (Department of Chemistry and Biochemistry, The Ohio State University), Paul Gollnick (Department of Biological Sciences, State University of New York at Buffalo, NY), 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:
Allostery is a ubiquitous mechanism for regulation of cellular processes allowing fine tuning of the cellular response to environmental cues. Despite the importance of allostery, we have limited understanding of the mechanism(s) by which activator ligands alter the properties of effector macromolecules. To better understand how allostery works we use native mass spectrometry (MS) to monitor allosterically-coupled population shifts in the model allosteric effector protein TRAP (trp RNA-binding attenuation protein) from Bacillus. TRAP forms a cyclic undecameric ring with 11 binding sites for the ligand tryptophan (Trp). When there is abundant Trp, up to 11 ligands bind to TRAP, altering its affinity towards its reporter ligand, a specific sequence of RNA of the trp operon, which encodes a series of Trp biosynthesis genes, resulting in transcription attenuation. It remains unclear how many Trp ligands must bind in order to activate TRAP, or how Trp and RNA binding are allosterically coupled. The allosteric coupling between Trp and RNA can be directly quantified from the population shift induced by the presence of RNA. We used a high-resolution Orbitrap mass spectrometer to measure nTrp:TRAP11 populations across a range of Trp concentrations in the absence and presence of RNA. Native mass spectra show that in the absence of RNA, Trp binding to TRAP follows a mostly Gaussian distribution, indicating little cooperativity between the Trp ligands themselves. In the presence of RNA, TRAP-RNA complexes show a skewed enrichment in Trp ligands, while the RNA-free protein exhibits a depletion of Trp. The population shifts illustrate the reciprocal nature of heterotopic allosteric effects: if Trp binding to TRAP promotes its binding to RNA, then the presence of RNA must promote the binding of Trp. This insight provides a solid framework for understanding how population shifts result in regulation in this and a wide range of similar allosteric switches.
Keywords: native mass spectrometry, RNA protein complexes