B. Imperiali J.J. Ottesen Authors' af®liations: B. Imperiali and J.J. Ottesen, Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Correspondence to: Barbara Imperiali Department of Chemistry Massachusetts Institute of Technology Cambridge MA 02139 USA Dates: Received 23 September 1998 Accepted 30 March 1999 To cite this article: Imperiali, B. & Ottesen, J.J. Uniquely folded mini-protein motifs. J. Peptide Res., 1999, 54, 177±184 Copyright Munksgaard International Publishers Ltd, 1999 ISSN 1397±002X Uniquely folded mini-protein motifs Key words: mini-protein motif; protein design; helix bundle; Betanova; BBA motif; charybdotoxin Abstract: Mini-proteins containing fewer than 40 amino acids provide simple model systems for studying protein folding and stability as well as serving as scaffolds for the rational design of new functional motifs. This article reviews current progress on the design and characterization of discretely folded mini-protein motifs. The past decade has witnessed signi®cant progress in the ®eld of protein design (1). Although it is not yet possible to exactly predict the native, folded structure of a protein from the linear sequence of amino acids that de®nes it, the rules for protein secondary and tertiary structure are becoming suf®ciently sophisticated that they now serve as an excellent guide for the design and redesign of small folded polypeptide motifs. In recent years, interest has focused on identifying the lower size limit of an independently folded polypeptide motif with supersecondary or tertiary structure. Approaching this lower size limit is valuable for the design of folded mini-motifs because small systems are the most readily amenable to structural analysis using contemporary biophysical methods, thus contributing to the successful execution of iterative design and synthesis approaches. This article will focus on the design and redesign of mini- protein motifs, which are de®ned as monomeric polypep- tides exhibiting discrete supersecondary structure in fewer than 40 amino acid residues and which fold with minimal assistance from cross-linking sites such as disul®des or metal binding centers. These motifs present highly simpli- ®ed model systems for studying protein folding and the stabilizing effects of speci®c residues (2±4). In addition, the 177