Employing directed evolution for the functional analysis of multi-specific proteins Maxim Levin a , Dotan Amar a , Amir Aharoni a,b,⇑ a Department of Life Sciences, Ben-Gurion University of the Negev, Be’er Sheva 84105, Israel b National Institute for Biotechnology in the Negev (NIBN), Ben-Gurion University of the Negev, Be’er Sheva 84105, Israel article info Article history: Available online 27 April 2013 Keywords: Multi-specific proteins Directed evolution Protein-networks abstract Multi-specific proteins located at the heart of complex protein–protein interaction (PPI) networks play essential roles in the survival and fitness of the cell. In addition, multi-specific or promiscuous enzymes exhibit activity toward a wide range of substrates so as to increase cell evolvability and robustness. How- ever, despite their high importance, investigating the in vivo function of these proteins is difficult, due to their complex nature. Typically, deletion of these proteins leads to the abolishment of large PPI networks, highlighting the difficulty in examining the contributions of specific interactions/activities to complex biological processes and cell phenotypes. Protein engineering approaches, including directed evolution and computational protein design, allow for the generation of multi-specific proteins in which certain activities remain intact while others are abolished. The generation and examination of these mutants both in vitro and in vivo can provide high-resolution analysis of biological processes and cell phenotypes and provide new insight into the evolution and molecular function of this important protein family. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction For many years, specificity has been considered as an essential property of proteins to enable their function in the complex cellu- lar environment. However, over the past two decades, this hypoth- esis has been challenged by many studies showing that low or multi-specificity in proteins is far from a rare phenomenon. Moreover, this property was shown to be universal for molecular recognition and was detected in many proteins participating in protein–protein, protein–peptide and protein–DNA interactions. The identification of multi-specific proteins participating in pro- tein–protein interaction (PPI) networks has been systematically investigated by several high-throughput experiments aimed at identifying all possible PPI in several different organisms. 1–3 These studies showed that while most proteins have only two–three interaction partners, a small but significant number of proteins can interact with more than 10 partners. In contrast to the identi- fication of proteins participating in multiple PPIs, the systematic identification of multi-specific enzymes is extremely difficult, due to the lack of a general screening approach for the detection of multiple catalytic reactions mediated by individual enzymes. Still, the abundance of multi-functional Escherichia coli enzymes was recently examined using a comprehensive bioinformatics ap- proach. This study found that around 40% of the E. coli metabolic enzymes are multi-specific, catalyzing 65% of metabolic reactions in the cell. 4 Further studies investigating the properties of the mul- ti-specific proteins showed that they are more conserved than are specific proteins and tend to be essential for organism viability. 5,6 In terms of cellular function, these multi-specific proteins play essential roles in enabling the function of complex cellular net- works by regulating the activity of numerous network proteins. 7,8 Despite the importance of multi-specific proteins for the sur- vival of organisms, the functional roles of these proteins in the cel- lular environment are difficult to investigate, due to their complex properties. Currently, the in vivo function of proteins is mainly examined following deletion of the gene encoding a target protein or a loss of function mutation. These modified strains are subse- quently examined for global cell phenotypes, including effects on viability, growth rate and sensitivity to a variety of chemical agents, or for defects in specific cellular processes, such as cell cy- cle progression, transcription regulation and protein folding. 9,10 In the case of multi-specific proteins, deletion of the encoding gene can lead to non-viability as the result of a complete disruption of the multiple interactions/catalytic activities of the protein, leading to the abolishment of large PPI networks. Thus, this all-or-none ap- proach prevents detailed examination of the contribution of spe- cific interactions or catalytic activities to complex biological processes and cell phenotypes. High-resolution analysis of the importance of specific interac- tions/activities of multi-specific protein in promoting biological processes can be performed using engineered mutants in which 0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmc.2013.04.052 ⇑ Corresponding author. Tel.: +972 8 6472645; fax: +972 8 6479218. E-mail address: aaharoni@bgu.ac.il (A. Aharoni). Bioorganic & Medicinal Chemistry 21 (2013) 3511–3516 Contents lists available at SciVerse ScienceDirect Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc