Poster abstracts

Poster number 161 submitted by Haoyun Yang

Nearest-neighbor cooperativity in the Bacillus ring-shaped trp RNA binding attenuation protein (TRAP) from protein engineering and cryo-EM

Haoyun Yang (Department of Chemistry and Biochemistry, The Ohio State University, Columbus, Ohio, USA), Weicheng Li, Andrew Norris, Vicki H. Wysocki, Mark P. Foster (Department of Chemistry and Biochemistry, Resource for Native Mass Spectrometry Guided Structural Biology, The Ohio State University, Columbus, Ohio, USA), Katie Lichtenthal, Skyler Kelly (Department of Biological Sciences, University at Buffalo, State University of New York, Buffalo, New York, USA.), Paul Gollnick (Department of Biological Sciences, University at Buffalo, State University of New York, Buffalo, New York, USA.), Vicki H. Wysocki, Mark P. Foster (Department of Chemistry and Biochemistry, Resource for Native Mass Spectrometry Guided Structural Biology, The Ohio State University, Columbus, Ohio, USA),

Abstract:
Homotropic cooperativity is a widespread mechanism for regulation in biological systems; the sigmoidal curve for oxygen binding to hemoglobin is a common pedagogical example 1. Homotropic cooperativity is particularly common in proteins that form symmetric homo-oligomeric assemblies whose biological activity is modulated by the binding of ligands 2,3. In such systems, even though the ligand binding sites are chemically identical, the affinity of a site for its ligand depends on whether other binding sites are already occupied. For macromolecules that arrange binding sites in a circular lattice a plausible mechanistic model attributes the cooperative interactions as arising from neighboring binding sites (also known as the nearest-neighbor effect)1. The affinity difference arises from allosteric interactions between adjacent ligand-binding sites that alter the free energy of ligand binding 4,5. The thermodynamic changes may arise from structural changes that are communicated between neighboring sites. Using native mass spectrometry (nMS) and isothermal titration calorimetry (ITC), we show that experimentally observed homotropic cooperativity in the dodecameric ring-forming gene regulatory protein TRAP from Bacillus halodurans (Bha) can be explained using a nearest-neighbor interaction model. To test this model, we used protein engineering to knock out ligand binding capabilities of alternating sites. We then apply cryogenic electron microscopy (CryoEM) to examine the structural basis of the proposed nearest-neighbor effect

References:
1.Stefan, Melanie I, and Nicolas Le Novère. “Cooperative binding.” PLoS computational biology vol. 9,6 (2013): e1003106.
2.Goodsell, D S, and A J Olson. “Structural symmetry and protein function.” Annual review of biophysics and biomolecular structure vol. 29 (2000): 105-53.
3.André, Ingemar et al. “Emergence of symmetry in homooligomeric biological assemblies.” Proceedings of the National Academy of Sciences of the United States of America vol. 105,42 (2008): 16148-52.
4.Brown, Alan. “Analysis of cooperativity by isothermal titration calorimetry.” International journal of molecular sciences vol. 10,8 3457-77. 4 Aug. 2009,
5.Ihms, Elihu C et al. “Mechanistic Models Fit to Variable Temperature Calorimetric Data Provide Insights into Cooperativity.” Biophysical journal vol. 112,7 (2017): 1328-1338.

Keywords: Protein engineering , Cryo-EM , native mass spectrometry