Carbon-Detected NMR Studies of Intrinsically Disordered Protein Post-Translational Modification

Carbon-Detected NMR Studies of Intrinsically Disordered Protein Post-Translational Modification

Access to the genetic information encoded in DNA must be both rapid and under tight control so that a cell can readily adapt to its environment, or rapidly respond to growth signals. One regulatory mechanism involves the use of proteins that have highly flexible regions that can be chemically modified in response to signals. These chemical modifications impact how these proteins associate with, or access, DNA and thus determine whether genetic information is accessible and expressed or hidden and suppressed. Thus, chemical modifications of these highly flexible proteins can exert timely control over cellular responses, acting to toggle their targets between active and inactive states and allowing the cell to quickly mobilize the resources it requires at the moment. Highly flexible regions are difficult to observe with current technologies; this fact significantly limits our understanding of how they function. The investigator will push the boundaries of current technologies and explore the structural consequences of chemical toggles of highly flexible proteins that control the expression of genetic information. This program addresses a significant gap in our knowledge of how gene expression is regulated. The research will train junior scientists at multiple education levels to seek fundamental insight into the biochemistry of biological processes using the principles and quantitative laws from the physical sciences. Among other outreach efforts, the investigator will bring middle school students from underprivileged backgrounds to Penn State to demonstrate the opportunities that arise from higher education in science and engineering fields.

To achieve this project's objectives, new experimental methodology will be implemented for nuclear magnetic resonance (NMR) spectroscopy of highly flexible proteins. While protons are relatively easy to observe by NMR, the PI has shown that the comparatively more challenging direct-detection of carbon yields more quantitative and complete information for intrinsically disordered polypeptides. Recently, the PI used this technique to demonstrate how serine phosphorylation of RNA polymerase II provides a structural switch that plays a role in regulating the critically important enzyme. The PI will now broaden the diversity of the highly flexible proteins that can be studied with carbon-detected NMR. Specifically, methods will be developed to overcome a current blind-spot for the aromatic amino acids tyrosine and phenylalanine. Second, the project will extend these studies to address the structural consequences of threonine phosphorylation. Finally, proteins that acquire phosphate in response to cellular and environmental signals often also acquire methyl groups. These modifications can exert combinatorial control, often in competition. Therefore, experiments will be performed to test the hypothesis that addition of methyl groups to the amino acid lysine also induces structural transitions, akin to those the PI has observed in association with phosphate incorporation. As a result, this research and associated training activities will yield fundamental, molecular level insights into gene regulation.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.