Protein circular permutation

Nagai, T., Yamada, S., Tominaga, T., Ichikawa, M. and Miyawaki, A. (2004). Expanded dynamic range of fluorescent indicators for Ca2+ by circularly permuted yellow fluorescent proteins. Proc. Natl. Acad. Sci. USA 101, 10554-9. 

The principle of in vivo cyclization is based on the circular permutation of precursor proteins containing an intein (Fig. 1.6 C) [74, 75, 80, 81]. A naturally occurring split intein, DnaE from Synechocystis sp. PCC6803, was first successfully used for cyclization. However, similarly to the IPL/EPL approach, a mixture of linear and circular forms is obtained, presumably because of hydrolysis of an intermediate [73, 75]. On the other hand, artificially split inteins such as Pl-Pful, DnaB, and the RecA intein have been successfully applied for in vivo cyclization, and only circular forms were observed [80-82], suggesting that the circular permutation approach is more suitable for cyclization. Compared to the IPL/EPL or the TWIN system, in vivo cyclization does not require any external thiol group for cyclization, similarly to protein ligation with split inteins. Moreover, there are no undesired products, such as linear forms or polymers, originating from intermolecular reactions. 

Potato type II (Potll) inhibitors are disulfide-rich peptides of approximately 50 amino acids in size. They were first discovered in leaves, seeds, and other organs of Solanaceae and are a source of much interest as plant defense proteins. Recently, Barta et analyzed expressed sequence tag (EST) and genomic data and discovered 11 genes that code for Potll inhibitors in various monocotyledonous and dicotyledonous plants. Potll inhibitors are expressed as large precursor proteins that contain up to eight sequence repeats of the inhibitor precursor. In one particularly fascinating case from the ornamental tobacco (N. data), the precursor adopts a circular permuted structure.Barta et al. observed that genes outside the Solanaceae family seem... 

Circularly permuted proteins 586, 587 Cloning genes 410-415 Clostripain 482... 

In proteins with a symmetric structure, circular permutation can account for the shift of active-site residues over the course of evolution. A very good model of symmetric proteins are the (/Ja)8-barrel enzymes with their typical eightfold symmetry. Circular permutation is characterized by fusion of the N and C termini in a protein ancestor followed by cleavage of the backbone at an equivalent locus around the circular structure. Both fructose-bisphosphate aldolase class I and transaldolase belong to the aldolase superfamily of (a/J)8-symmetric barrel proteins both feature a catalytic lysine residue required to form the Schiff base intermediate with the substrate in the first step of the reaction (Chapter 9, Section 9.6.2). In most family members, the catalytic lysine residue is located on strand 6 of the barrel, but in transaldolase it is not only located on strand 4 but optimal sequence and structure alignment with aldolase class I necessitates rotation of the structure and thus circular permutation of the jS-barrel strands (Jia, 1996). 

Fig. 1. Schematics of evolutionary mechanisms of domain swapping in nature. Multifunctional proteins arise from the fusion of the genes coding for individual enzymes. Often the individual domains of multifunctional proteins catalyze successive steps in metabolic pathways. In tandem duplication, a gene is duplicated and the 3 end of one copy is fused in-frame to the 5 end of the second copy. In domain recruitment, a functional unit (whole gene or gene fragment) from one gene is either inserted within or fused to an end of a second gene. Circular permuted genes are believed to arise via tandem duplication followed by introduction of new start and stop codons (Ponting el at, 1995).

Even greater topological changes in GFP have been performed by random circular permutation (Graf and Schachman, 1996) in order to create new N- and C- termini for end-to-end fusion with other genes (Baird et al., 1999). Ten nontrivial fluorescent circular permutations of GFP were found that had altered pIQ values and orientation of the chromophore with respective to its N- and C-termini. The systematic identification of sites for circular permutation in GFP also identifies plausible sites for insertions of other proteins into GFP. This work speaks strongly about the potential of random circular permutation for protein... 

Heinemann, U., and Hahn, M. (1995). Circular permutation of polypeptide chains implications for protein folding and stability. Prog. Biophys. Mol. Biol, 64, 121-143. 

Hennecke.J., Sebbel, P., and Glockshuber, R. (1999). Random circular permutation of DsbA reveals segments that are essential for protein folding and stability. J. Mol. Biol, 286, 1197-1215. 

Lindqvist, Y., and Schneider, G. (1997). Circular permutations of natural protein sequences structural evidence. Curr. Opin. Struct. Biol, 7, 422—427. 

Circular permutation of a protein results in the relocation of its N- and C-termini within the existing structural framework. Initiated by a tandem duplication of a precursor gene, one mechanistic model proposes an in-frame fusion of the original termini, followed by the generation of a new start codon in the first repeat and a termination site in the second. In support of the model, tandem duplications are observed in prosaposins [29] and DNA methyltransferases [30], both genes for which circular per-mutated variants are also known. 

Evidence for domain recruitment has been identified in a wide variety of proteins [47], mechanistically ranging from simple N- or C-terminal fusion to multiple internal insertions and possibly circular permutations [48]. A recent analysis of proteins in the protein structure database (PDB) has further indicated that structural rearrangements as a result of domain shuffling have significantly contributed to today s functional diversity [49]. A brief overview of the various modes of domain recruitment and their effects on function, is presented on examples of /lex-barrel structures. 

Figure 1. General PCR protocol for the creation of circularly permuted proteins.

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