Notch1 EGF repeats
Investigating the roles of glycosylation and calcium in the structure and dynamics of Notch1
The long-term goal of this project is to predict the structure and dynamics of the tandem epidermal growth factor-like (EGF) repeats based on calcium binding and glycosylation. It is well-known that strong calcium binding sites between EGF repeats align the domains in a rigid rod-like orientation, but little is known about the effects of weak calcium binding or glycosylation on EGF structure and dynamics. Our hypotheses are that calcium binding and glycosylation regulate protein function through cooperative conformational changes. Our target for this project is the Notch1 protein, specifically the extracellular Abruptex domain involved in ligand recognition and activation. Notch1 proteins are transmembrane receptors that receive intercellular signals and determine cell specification by binding the ligands, Delta or Serrate/Jagged, on signal-sending cells. The extracellular domains of Notch1, Delta, and Serrate/Jagged consist of multiple EGF repeats. The Abruptex domain of Notch1 consists of six EGF repeats (EGF24-29), is highly glycosylated and flexible, and negatively regulates Notch1 activation through interactions with the ligand binding domain (EGF11-12). Currently, we are working toward the high resolution structures of glycosylated EGF27 and EGF26. Our collaborator, Dr. Robert Haltiwanger from the University of Georgia, Complex Carbohydrate Research Center, supports this research by providing assistance with protein glycosylation.
Justin Grennell and Kendra Jenkins are the lead graduate students on this project.
This project was supported by NSF CHE-1413576.
Related publications
- J. A. Grennell and M. A. Macnaughtan, Backbone and side-chain assignments of mouse NOTCH1 epidermal growth-factor-like repeat 27. In preparation for Biomolecular NMR Assignments.
- J. A. Grennell, K. D. Jenkins, H. Zhong, A. Paudyal, K. B. Luther, R. S. Haltiwanger, M. A. Macnaughtan, Expression, purification, and glycosylation of epidermal growth factor-like repeat 27 from mouse NOTCH1. Protein Expression and Purification, 2020, 174, 105681-105692.
- H. Takeuchi, R. C. Fernández-Valdivia, D. S. Caswell, A. Nita-Lazar, S. Kakuda, N. A. Rana, T. Garner, T. Weldeghiorghis, M. A. Macnaughtan, H. Jafar-Nejad, and R. S. Haltiwanger,* Rumi functions as both a protein O-glucosyltransferase and a protein O-xylosyltransferase. Proceedings of the National Academy of Sciences, 2011, 108(40), 16600-16605.