Destabilizing an interacting motif strengthens the association of a designed ankyrin repeat protein with tubulin.

ABSTRACT: Affinity maturation by random mutagenesis and selection is an established technique to make binding molecules more suitable for applications in biomedical research, diagnostics and therapy. Here we identified an unexpected novel mechanism of affinity increase upon in vitro evolution of a tubulin-specific designed ankyrin repeat protein (DARPin). Structural analysis indicated that in the progenitor DARPin the C-terminal capping repeat (C-cap) undergoes a 25° rotation to avoid a clash with tubulin upon binding. Additionally, the C-cap appears to be involved in electrostatic repulsion with tubulin. Biochemical and structural characterizations demonstrated that the evolved mutants achieved a gain in affinity through destabilization of the C-cap, which relieves the need of a DARPin conformation

SUBMITTER: Ahmad S 

PROVIDER: S-EPMC4933879 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature