title: Studies of the ankyrin repeats of the Drosophila melanogaster Notch receptor. 2. Solution stability and cooperativity of unfolding.
abstract: To define the boundaries of the Drosophila Notch ankyrin domain, examine the effects of repeat number on the folding of this domain, and examine the degree to which the modular architecture of ankyrin repeat proteins results in modular stability, we have investigated the thermodynamics of unfolding of polypeptides corresponding to different segments of the ankyrin repeats of Drosophila Notch. We find that a polypeptide containing the six previously identified ankyrin repeats unfolds cooperatively, but is of modest stability. However, inclusion of a putative seventh, C-terminal ankyrin sequence doubles the stability of the Notch ankyrin domain (a 1000-fold increase in the folding equilibrium constant), indicating that the seventh ankyrin repeat is an important part of the Notch ankyrin domain, and demonstrating long-range interactions among ankyrin repeats. This putative seven-repeat polypeptide also shows increases in enthalpy, denaturant dependence (m-value), and heat capacity of unfolding (DeltaC(p)()) of around 50% each, suggesting that deletion of the seventh repeat results in partial unfolding of the sixth ankyrin repeat, consistent with spectroscopic and hydrodynamic data reported in the preceding paper [Zweifel, M. E., and Barrick, D. (2001) Biochemistry 40, 14344-14356]. A polypeptide consisting of only the five N-terminal repeats has stability similar to the six-repeat construct, demonstrating that stability is distributed asymmetrically along the ankyrin domain. These data are consistent with highly cooperative two-state folding of these ankyrin polypeptides, despite their modular architecture.
authors.affiliation: T. C. Jenkins Department of Biophysics, The Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, USA.
authors.forename: M E
authors.initials: ME
authors.lastname: Zweifel
authors.forename: D
authors.initials: D
authors.lastname: Barrick
mesh_terms: D000818:Animals; D017089:Ankyrin Repeat; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000737:chemistry*; D004795:Enzyme Stability; D008565:Membrane Proteins / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D051880:Receptors, Notch; D013050:Spectrometry, Fluorescence; D013223:Statistics as Topic; D013696:Temperature; D014508:Urea
chemical_list: D029721:Drosophila Proteins; D008565:Membrane Proteins; C497759:N protein, Drosophila; D051880:Receptors, Notch; D014508:Urea