The development of biomimetic routes to cyclic proteins and peptides is an area of great current interest. Cyclization confers conformational constraints that are thought to result in higher biological potency and stability of the resulting protein or peptide. This strategy has been utilized by nature, with cyclosporin A and gramicidin S being two well-studied examples of naturally occurring cyclic peptides (Kahan 1982; Polin and Egorov 2003).

1.1 Naturally Occurring Cyclic Peptides

Marine sponges have proven to be a rich source of cyclic peptides that are wide ranging in structure and biological activity (Milanowski et al. 2004; Schmidt et al. 2004), many of which contain non-proteinogenic amino acids and polyketide-derived moieties. Fungi isolated from various marine organisms have also yielded several biologically active cyclic peptides (Abarzua and Jakubowski 1995; Bringmann et al. 2004).

Naturally occurring cyclic peptides can be divided in two classes. Cyclic non-ribosomal peptides (such as the immunosuppressant cyclosporin A) are synthesized enzymatically by the ordered condensation of monomer building blocks to produce a linear peptide that is cyclized by a thioester domain at the C-terminus at the end of the biological process (Kohli and Walsh 2003).

The structures of a different second class of naturally occurring cyclic peptides have been reported recently (Trabi and Craik 2002; Craik et al. 2003). Present in a variety of organisms, they are distinct from non-ribosomal cyclic

A. Tavassoli, T.A. Naumann, S.J. Benkovic

Department of Chemistry, Pennsylvania State University, University Park, Pennsylvania 16802, USA

Nucleic Acids and Molecular Biology, Vol. 16 Marlene Belfort et al. (Eds.) Homing Endonucleases and Inteins © Springer-Verlag Berlin Heidelberg 2005

peptides in that they are produced via transcription and translation of genes and have a well-defined three-dimensional structure. They differ from linear peptides only in their post-translational modification to join the N- and C-termini. In bacteria the currently known cyclic peptides of this class range from 21 to 78 amino acids and vary considerably in primary sequence and structure; no homology is evident between the amino acids involved in the de novo peptide bond or the flanking regions (Martinez-Bueno et al. 1994; Sol-biati et al. 1999). Their biosynthesis involves diverse auxiliary proteins and a common mechanism of cyclization is thought unlikely (Craik et al. 2003).

In plants, an interesting group of cyclic peptides known as the cyclotides (Craik et al. 1999) exist that contain six conserved cysteine residues that form a series of disulfide bonds in their protein core (Fig. 1; Rosengren et al. 2003). The multiple internal disulfide bonds confer interesting structural characteristics upon the cyclotides resulting in twisted, knotted, and ringed cyclic peptides (Rosengren et al. 2003). The resulting structural rigidity is thought to be responsible for their resistance to proteolysis and stability; cyclotides have also been found to have a range of bio activities, ranging from anti-HIV (Daly et al. 1999) to potent insecticides (Jennings et al. 2001).

The advantage of cyclic over linear peptides is demonstrated by the naturally occurring antibacterial mammalian peptide rhesus theta defensin (RTD-1), biosynthesized by ligation of two linear peptides. RTD-1 in its native cyclic form has been shown to have three times the antibacterial activity of the linear synthetic analog (Tang et al. 1999). This difference has been attributed to the extra stability provided by the cyclic form in vivo and not structural changes upon cyclization.

Fig. 1. The structure of Kalata Bl. The three internal disulfide bonds are highlighted

1.2 Intein-Mediated Cyclization

Utilization of intein-based methods has resulted in the use of bacterial protein expression for the synthesis of cyclic versions of linear proteins. Cyclization has been shown to offer two main advantages, increased in vivo stability and biological activity, as a result of resistance to degradation by proteases and decreasing conformational flexibility over the linear protein counterpart (Hruby 1982; Hruby et al. 1990). Here, we describe recent developments in in-tein-based methodology for the biosynthesis of cyclic peptides and cyclization of linear proteins. Although there are several examples of recently developed methods and many studies into the use of cyclic peptides as pharmaceutical agents, it should be noted that the field is still in relative infancy.

Protein splicing is a self-catalyzed post-translational process in which an intervening internal sequence (intein) excises itself out of a precursor polypeptide resulting in the concomitant linkage of the flanking sequences (exteins) by a native peptide bond. Over 100 inteins have been identified in bacteria and unicellular eukaryotic organisms (Perler 2000); whilst they vary in size (134-600 amino acids), a set of highly conserved residues exist at the splicing junction (Pietrokovski 1994,1998; Perler et al. 1997). Inteins generally begin with serine or cysteine and end in asparagine; the C-terminal of the intein is always followed by a serine, threonine or cysteine. There is little homology in the extein sequence thus the native exteins can be replaced by a foreign sequence without a dramatic adverse effect on splicing and cleavage. Naturally occurring inteins can be subdivided into three groups: those containing a homing endonuclease between the splicing domains, those lacking a homing endonuclease region, and those in which the splicing domain is split, the so-called trans-inteins.

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