Poster abstracts
Poster number 28 submitted by Weicheng Li
Insights into the Homotropic Allostery of the Oligomeric Trp- and RNA-binding protein TRAP via Mechanistic Modelling and Protein Engineering
Weicheng Li, Elihu C. Ihms, Ian R. Kleckner, Melody L. Holmquist (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH43210), Paul Gollnick (Department of Biological Sciences, State University of New York at Buffalo, Buffalo, NY14260), Mark P. Foster (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, OH43210)
Abstract:
Allostery, the process of conveying the effect of binding at one site to another functional site in macromolecules, regulates virtually every biological process, such as transport, transcription, and enzyme activity. However, the limited understanding about how allostery is realized at a microscopic level—the thermodynamic consequences of the allosteric propagations—prevents the understanding of the macroscopic currents. TRAP (trp RNA-binding Attenuation Protein) is a homo-undecamer whose RNA binding activity is regulated by binding of L-tryptophan (Trp) to its eleven identical binding sites. The Trp binding events are thought to stabilize the RNA-binding state of TRAP and favor RNA binding. Trp binding events show homotropic allostery and cooperativity clearly informed by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) experiments. By fitting temperature-dependent ITC data using a nearest-neighbor (NN) statistical thermodynamic model, Trp binding to Bst TRAP A28I, we quantify the microscopic contributions of Trp binding to the affinity at neighboring sites. The NN model provides the microscopic descriptions of how Trp-binding events are energetically affected by the occupancy of neighboring sites. The microscopic descriptions can be extrapolated to predict macroscopic binding behavior. However, the parameters of the NN model are all obtained by global fitting. To directly determine the parameters experimentally, we are engineering the protein to eliminate the effect of nearest neighbor interactions, which would allow the direct determination of intrinsic binding affinity of Trp to TRAP protein, optimizing the NN model. It is expected to accurately describe the energetic interactions of homotropic allostery in the RNA-binding protein TRAP and it may give the hint of how does Trp stabilize the RNA-binding.
Keywords: Allostery, statistical thermodynamic model, Protein Engineering