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Contents : METHODS IN MOLECULAR BIOLOGY 340 Protein Design Methods and Applications Edited Edited by Raphael Guerois Manuela L pez de la Paz Design of Repeat Proteins 151 7 Consensus Design as a Tool for Engineering Repeat Proteins Tommi Kajander Aitziber L. Cortajarena and Lynne Regan Summary Repeat proteins were first identified because of their unusual primary structure in which short amino acid sequences typically between 20 and 40 residues are repeated in tandem often many times. After identification at the sequence level the three-dimensional structures of representatives from several classes (e.g. ankyrin tetratricopeptide leucine rich repeat) have been solved. The structures indeed reveal unusual nonglobular structures a linear string of the tandem motifs. Perhaps because of the large surface area that is presented as a consequence of such elongated structures repeat domains are often involved in mediating protein protein interactions. Here we describe methods of consensus-based design and engineering of repeat proteins. We pay particular attention to the attributes of repeat proteins that make them well-suited to such approaches. In addition we discuss practical issues related to producing and characterizing such designed proteins. We use the tetratricopeptide repeat which is well-studied in our group to illustrate many ideas but also draw comparisons to other work on repeat proteins where relevant. Key Words: Repeat protein tetratricopeptide repeat TPR protein engineering protein design binding domain consensus sequence. 1. Introduction Protein design initially concentrated on understanding the structural determinants and folding of small globular all- helical or all- sheet proteins (1 4). More recently various modeling strategies which incorporate exhaustive conformational searches have been applied to a variety of folds (e.g. refs. 5 8). Another approach to structural design the design of proteins based on the amino acid consensus from multiple sequence alignments emerged in the 1990s. The notion was to choose the most probable amino acid for each position by analyzing the statistics of occurrence of different amino acids at each position in a sequence alignment of as many related proteins as possible. The From: Methods in Molecular Biology vol. 340: Protein Design: Methods and Applications Edited by: R. Guerois and M. L pez de la Paz Humana Press Inc. Totowa NJ 151 152 Kajander Cortajarena and Regan first successful application of this approach was the design of a consensus zincfinger peptide by Berg and colleagues. The peptide they designed bound metal ions and adopted the typical zinc finger fold (9). The underlying hypothesis of this approach was that stabilizing amino acids should be present in the sequence data with high frequency and thus this strategy should not only specify the protein fold but also yield more stable proteins. This was indeed the case for their zinc-finger peptide designs and has proven true for many subsequent consensus-based designs. Variations on consensus-based engineering have since been applied in several systems as a method to enhance stability for example in immunoglobulin variable domains (10) phytases (11) and SH3domains (12). More recently the consensus design approach has been applied to nonglobular repeat motifs such as the tetratricopeptide repeat (TPR) ankyrin and leucine-rich repeat (LLR) with slightly differing strategies in several laboratories (13 16). Such motifs contain a repeating unit of amino acid sequence (34 amino acids in TPR proteins and 33 amino acids in ankyrins for example) which can occur in tandem arrays of varying length in different proteins. Most characteristically the repeat proteins are open-ended structures which can have varying numbers of repeat units stacked in tandem arrays forming a domain (Fig. 1). Different repeat motifs form distinct three-dimensional structures. In most cases tandem repetition of the motif gives rise to elongated structures (17) but with different twists and curvatures (18). The TPRs for example form right-handed superhelical structures. Repeat proteins are common particularly in the higher organisms comprising 5.3% of the metazoan proteins annotated in the SwissProt database (17). Also LLR ankyrin and TPR repeats are among the 20 most common motifs found in PFAM (i.e. protein families) (http:// www.sanger.ac.uk/Software/Pfam/browse/top twenty.shtml). Thus far in all known examples the nonglobular repeat domains appear to be involved in macromolecular interactions although the exact function and identity of the binding partner is not known for the majority of repeat proteins. In this chapter we describe methods used in our laboratory to design synthetic consensus TPR proteins and discuss other developments in protein engineering studies of repeat proteins where applicable. 2. Materials 2.1. Computer Calcula

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