The tolerance of a modular protein to duplication and deletion of internal repeats

Ankyrin repeat polypeptides contain repeated structural elements that pack to produce modular architectures lacking in close contacts between distant segments of the polypeptide chain. Despite this lack of sequence-distant contacts, ankyrin repeat polypeptides have been shown to fold in a cooperative manner. To determine the distance over which cooperative interactions can be propagated in a repeat protein, and to investigate the tolerance to internal duplication and deletion of modules, we have constructed a series of ankyrin repeat variants of the Notch ankyrin domain in which repeat number is varied by duplication and deletion of internal repeats. A construct with two copies of the fifth ankyrin repeat shows a modest increase in stability compared to the parent construct and retains apparent two-state unfolding behavior. Although constructs containing three and four copies of the fifth repeat retain this increased resistance to urea, they exhibit broad, multi-state unfolding transitions compared to the parent construct. For the Notch ankyrin domain, these larger constructs may represent a limit beyond which full cooperativity cannot be maintained. Deletions of internal repeats from the Notch ankyrin domain significantly destabilize the domain. This severe destabilization, which is larger than that resulting from end-repeat deletion, may arise from unfavorable interactions within the new non-native interfaces produced by internal repeat deletion. These results demonstrate both an asymmetry between the duplication and deletion of internal repeats, and a difference between deletion of internal and end-repeats, suggesting preferred mechanisms for evolution of repeat proteins.