Design of a Heterospecific, Tetrameric, 21-Residue Miniprotein with Mixed α/β Structure

Summary The study of short, autonomously folding peptides, or "miniproteins," is important for advancing our understanding of protein stability and folding specificity. Although many examples of synthetic α-helical structures are known, relatively few mixed α/β structures have been successfully designed. Only one mixed-secondary structure oligomer, an α/β homotetramer, has been reported thus far. In this report, we use structural analysis and computational design to convert this homotetramer into the smallest known α/β-heterotetramer. Computational screening of many possible sequence/structure combinations led efficiently to the design of short, 21-residue peptides that fold cooperatively and autonomously into a specific complex in solution. A 1.95 A crystal structure reveals how steric complementarity and charge patterning encode heterospecificity. The first- and second-generation heterotetrameric miniproteins described here will be useful as simple models for the analysis of protein-protein interaction specificity and as structural platforms for the further elaboration of folding and function.