Characterization of protein folding determinants for LIN-12/Notch-Repeats (LNRs) using human Notch1 LNRB as a model system

CHED 794

Sharline Madera, smadera@wellesley.edu and Didem Vardar-Ulu, dvardar@wellesley.edu. Department of Chemistry, Wellesley College, 21 Wellesley College Rd, Unit 7633, Wellesley, MA 02481
Notch proteins are a family of transmembrane glycoproteins that control differentiation in multicellular animals. Their highly conserved modular architecture contains three contiguous LIN-12/Notch-Repeats (LNRs), LNRA, LNRB and LNRC that maintain the receptor in its resting conformation prior to ligand binding. These ~35 residue long, cysteine rich repeats contain a characteristic arrangement of disulfide bonds and require Ca2+ for correct folding. In human Notch1, the LNRs are separated by two linkers, 10 and 5 residues long, respectively. In this work, we used LNRB from human Notch1 as a model system to define the minimum requirements for a correctly folded LNR. We determined that part of the N-terminal linker was indispensable and folding process was selective for Ca2+. We further investigated the impact of removing the first disulfide bond on LNRB folding to further compare it with LNRA from human Notch4, the only natural LNR known to possess two disulfide bonds.