Folding and design of ankyrin repeat proteins

Date of Completion

January 2004

Keywords

Chemistry, Biochemistry|Biophysics, General

Degree

Ph.D.

Abstract

The ankyrin repeat is one of the most frequently occurring amino acid repeats in nature. This 33-residue motif is typically arranged in tandem repeats that stack against one another to form the underlying scaffold for many protein-protein interactions. Despite the modular nature of ankyrin repeat domains, the bulk of equilibrium folding studies indicate that these repeats follow a two-state folding pathway rather than multi-state. Our studies on the cardiomyogenic hormone, myotrophin, support this conclusion. To determine the important positions for structure formation and create a generic molecular scaffold, statistical analysis of thousands of ankyrin repeats was used to design a consensus ankyrin repeat sequence. Proteins containing three and four identical consensus repeats (3ANK and 4ANK) were well-folded with high thermostability. High resolution X-ray crystallography showed these proteins to have the ankyrin repeat fold with a periodic network of hydrogen bonds. Our statistical analysis was also useful in engineering greater solubility of 4ANK at physiological pH as well as introducing tryptophans into 4ANK to characterize the folding pathway. These results clearly demonstrate that the ankyrin repeat consensus sequence contains all the necessary information to form the ankyrin repeat fold. The de novo designed ankyrin repeat proteins can serve as universal scaffolds on which to engineer specific protein binding interfaces and our folding and design work may serve as a general model for other repeat motifs. ^

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