Protein Design by Directed Evolution

Abstract

While nature evolved polypeptides over billions of years, protein design by evolutionary mimicry is progressing at a far more rapid pace. The mutation, selection, and amplification steps of the evolutionary cycle may be imitated in the laboratory using existing proteins, or molecules created de novo from random sequence space, as starting templates. However, the astronomically large number of possible polypeptide sequences remains an obstacle to identifying and isolating functionally interesting variants. Intelligently designed libraries and improved search techniques are consequently important for future advances. In this regard, combining experimental and computational methods holds particular promise for the creation of tailored protein receptors and catalysts for tasks unimagined by nature.

Establishing wild-type levels of catalytic activity on natural and artificial (βα) 8 -barrel protein scaffolds

“…Recent technical advances have allowed experimental and computational biochemists to alter the activity and stability of these exquisite molecular machines in a predefined manner, either by rational design or directed evolution (2)(3)(4)(5)(6). The results of such experiments help to understand the complex interplay between the structure and function of enzymes, and provide insights into the mechanisms by which they have evolved from less sophisticated precursors.…”

“…Specifically, the modular construction of (␤␣) 8 -barrels suggests that the mixing and matching of (␤␣) n entities might be a mechanism to generate stable proteins, on which novel catalytic activities can be established (20). Indeed, the striking internal 2-fold symmetry observed for both HisA and imidazole glycerol phosphate synthase (HisF), which catalyzes the subsequent reaction within histidine biosynthesis, suggests that these 2 enzymes are composed of 2 (␤␣) 4 -half barrel domains (21,22). The construction of the chimeric protein HisAF consisting of the 4 N-terminal (␤␣) 1-4 units of HisA and the 4 C-terminal (␤␣) [5][6][7][8] units of HisF provides further experimental support for this hypothesis.…”

Design, selection, and characterization of a split chorismate mutase

“…18,[22][23][24][36][37][38] Here we show for the first time the construction of a functional heterodimeric CM. Simply cleaving the dimer-spanning N-terminal helix of MjCM to yield a one-helix and a three-helix fragment abolishes enzyme function in vitro and in vivo.…”

“…18,[21][22][23][24] Insertion of short peptide sequences into the middle of the dimer-spanning N-terminal helix converts the natural homodimer into monomers (mMjCM), 18 trimers, 22 and hexamers. 22 The tolerance of the N-terminal helix to disruption between residues 21 and 22 also makes this an attractive cleavage site for the generation of a heterodimeric mutase [ Fig.…”

E

“…Examples are the evolutionary design of nucleic acid molecules (Joyce 2004;Klussmann 2006), proteins (Brakmann andJohnsson 2002;Jäckel et al 2008), and synthetic compounds (Wrenn and Harbury 2007). Quasispecies theory and the concept of error propagation in successive replication gave rise to new antiviral strategies known as lethal mutagenesis (Domingo et al 2008).…”