Binding of small molecules to cavity forming mutants of a de novo designed protein.

Department of Chemistry, Princeton University, Princeton NJ 08544-1009.

A central goal of protein design is to devise novel proteins for applications in biotechnology and medicine. Many applications, including those focused on sensing and catalysis, will require proteins that recognize and bind to small molecules. Here we show that stably folded a-helical proteins isolated from a binary patterned library of designed sequences can be mutated to produce binding sites capable of binding a range of small aromatic compounds. Specifically, we mutated two phenylalanine side chains to alanine in the known structure of de novo protein S-824 to create buried cavities in the core of this 4-helix bundle. The parental protein and the Phe?Ala variants were exposed to mixtures of compounds, and selective binding was assessed by saturation transfer difference (STD) NMR. The affinities of benzene and a number of its derivatives were determined by pulse field gradient spin echo (PFGSE) NMR, and several of the compounds were shown to bind the mutated protein with micromolar dissociation constants. These studies suggest that stably folded de novo proteins from binary patterned libraries are well-suited as scaffolds for the design of binding sites.

Copyright © 2011 The Protein Society.

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