title: Limits of cooperativity in a structurally modular protein: response of the Notch ankyrin domain to analogous alanine substitutions in each repeat.
abstract: To determine the limits of cooperativity in a structurally modular protein, we characterized the structure and stability of glycine variants of the ankyrin repeat domain from the Drosophila melangaster Notch receptor. The substitutions are of analogous alanine residues to glycine in each repeat, and allow the same perturbation to be examined at different positions in the protein. The ankyrin domain is insensitive to substitution in repeat one, suggesting that the first repeat is not fully-folded. Glycine substitutions in repeat two through seven are strongly destabilizing, but the variants retain their overall secondary and tertiary structures. Spectroscopic and calorimetric data are consistent with two-state unfolding transitions for the repeat-two through repeat-five glycine variants, and for the wild-type protein. These data indicate that, despite its modular structure, the Notch ankyrin domain unfolds as a cooperative unit consisting of the six C-terminal repeats, and that this cooperativity is maintained in the presence of severely destabilizing substitutions in the N-terminal and central repeats. In contrast, glycine substitution in repeat six leads to a multi-state unfolding transition, suggesting that the coupling that gives rise to long-range cooperativity in the wild-type protein may have a weak link in the C-terminal region. Such behavior is captured by a simple statistical thermodynamic model in which an unstable C-terminal region is coupled to a stable N-terminal region through a strongly stabilizing interface.
authors.affiliation: T. C. Jenkins Department of Biophysics, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA.
authors.forename: Christina Marchetti
authors.initials: CM
authors.lastname: Bradley
authors.forename: Doug
authors.initials: D
authors.lastname: Barrick
mesh_terms: D000409:Alanine; 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 / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011956:Receptors, Cell Surface / Q000737:chemistry*; D051880:Receptors, Notch; D013050:Spectrometry, Fluorescence; D013696:Temperature; D013816:Thermodynamics; D014508:Urea
chemical_list: D029721:Drosophila Proteins; D008565:Membrane Proteins; C497759:N protein, Drosophila; D011956:Receptors, Cell Surface; D051880:Receptors, Notch; D014508:Urea; D000409:Alanine