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 investigate the relationship between structure and stability in ankyrin repeat proteins, we have examined structural features of polypeptides from the Drosophila melanogaster Notch protein that contain five, six, and a putative seventh ankyrin repeat. Circular dichroism (CD) spectroscopy indicates that Notch ankyrin polypeptides of different length contain a significant amount of alpha-helix, indicating that a folded structure can be maintained despite the loss of individual ankyrin modules. However, the alpha-helical content of the construct with the putative seventh repeat is slightly higher than polypeptides containing fewer repeats, suggesting that the putative seventh repeat may help stabilize other parts of the ankyrin domain. Fluorescence spectroscopy indicates that the single tryptophan in the fifth ankyrin repeat is in a nonpolar environment and is shielded from solvent in all three constructs, 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 indicate that these ankyrin repeat polypeptides are monomeric. Sedimentation velocity studies indicate that each polypeptide exhibits significant axial asymmetry, consistent with the elongated structure seen for other for ankyrin repeat proteins. However, the degree of asymmetry is greatest for the construct containing six repeats, suggesting that in the absence of the putative seventh repeat, the sixth repeat is partly unfolded.
Zweifel
M E
ME
T. C. Jenkins Department of Biophysics, The Johns Hopkins University, Baltimore, Maryland 21218, USA.
Barrick
D
D
D000818:Animals; D017089:Ankyrin Repeat; D001665:Binding Sites; D002942:Circular Dichroism; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D051880:Receptors, Notch; D013050:Spectrometry, Fluorescence; D014461:Ultracentrifugation
D029721:Drosophila Proteins; D008565:Membrane Proteins; C497759:N protein, Drosophila; D051880:Receptors, Notch