Biological synthesis of circular polypeptides

Abstract

Here, we review the use of different biochemical approaches for biological synthesis of circular or backbone-cyclized proteins and peptides. These methods allow the production of circular polypeptides either in vitro or in vivo using standard recombinant DNA expression techniques. Protein circularization can significantly impact protein engineering and research in protein folding. Basic polymer theory predicts that circularization should lead to a net thermodynamic stabilization of a folded protein by reducing the entropy associated with the unfolded state. Protein cyclization also provides a valuable tool for exploring the effects of topology on protein folding kinetics. Furthermore, the biological production of cyclic polypeptides makes possible the production of cyclic polypeptide libraries. The generation of such libraries, which was previously restricted to the domain of synthetic chemists, now offers biologists access to highly diverse and stable molecular libraries for probing protein structure and function.

SCHEME 1.

Summary of methods employed for biological production of backbone-cyclized peptides. Left panel, these approaches rely on the ribosomal synthesis of a polypeptide precursor that undergoes cyclization mediated by inteins or proteases. Middle panel, shown are three-dimensional structures of three examples of naturally occurring circular peptides with potential biotechnological value. MCoTI-II and SFTI-1 are powerful protease inhibitors and are attractive scaffolds for the introduction of other biological activities. RTD-1 is a θ-defensin that has strong antimicrobial and anti-inflammatory properties. All have been successfully expressed in biological systems (see Table 1). Right panel, the potential biotechnological applications of circular polypeptides, which include pharmacological, agrochemical, and diagnostic applications, are summarized. Molecular structures were generated using the structures of MCoTI-II (Protein Data Bank code 1IB9), SFTI-1 (code 1JBN), and RTD-1 (code 1HVZ).

FIGURE 1.

Three most common approaches for biological production of backbone-cyclized polypeptides using EPL (A), PTS (B), and SML (C). I_C, C-intein; I_N, N-intein.