Studies of the ankyrin repeats of the Drosophila melanogaster Notch receptor. 1. Solution conformational and hydrodynamic properties

To gain insight into the structural basis for Notch signaling, and to inves tigate the relationship between structure and stability in ankyrin repeat p roteins, we have examined structural features of polypeptides from the Dros ophila melanogaster Notch protein that contain five, six, and a putative se venth ankyrin repeat. Circular dichroism (CD) spectroscopy indicates that N otch ankyrin polypeptides of different length contain a significant amount of a-helix, indicating that a folded structure can be maintained despite th e loss of individual ankyrin modules. However, the a-helical content of the construct with the putative seventh repeat is slightly higher than polypep tides containing fewer repeats, suggesting that the putative seventh repeat may help stabilize other parts of the ankyrin domain. Fluorescence spectro scopy indicates that the single tryptophan in the Fifth ankyrin repeat is i n a nonpolar environment and is shielded from solvent in all three construc ts, although slight differences in spectroscopic properties of the six- and five- repeat constructs are observed, indicating minor structural changes. Near-UV CD indicates that these ankyrin polypeptides contain a significant amount of fixed tertiary structure surrounding their aromatic side chains. Gel filtration chromatography and sedimentation equilibrium studies indica te that these ankyrin repeat polypeptides are monomeric. Sedimentation velo city studies indicate that each polypeptide exhibits significant axial asym metry, consistent with the elongated structure seen for other for ankyrin r epeat proteins. However, the degree of asymmetry is greatest for the constr uct containing six repeats, suggesting that in the absence of the putative seventh repeat, the sixth repeat is partly unfolded.