**5.1 Lacking co-structural model**

To introduce tailored functions into a protein scaffold is never an easy task. For its ultra stability and high diversity of function, there is no doubt that peptide with a CSαβ motif is a suitable scaffold to be engineered for biomedical applications. To date, there are only a few of successful cases are reported. Many structures of peptides with CSαβ motif have been solved and deposited in database, but knowledge about the motif is poor. In spite of hundreds of peptides with a CSαβ motif deposited in the database, it is hard to make conclusions relating to structures and biochemical functions of the motif. Key residues are

Multiple mutation also has been performed on the scaffold. Vital *et al* performed minimal residue substitutions on the charybodotxin (Chtx), an scorpion toxin containing CSαβ motif, to equip metal ion binding ability (Vita et al., 1995). Three residues, K27, M28 and R34, on the β sheet are substituted with histidines. The modified protein exhibts a chelatine property

Grafting epitopes with known function is a straightforward stratage for a protein to gain new function. Functional epitopes could be exchanged between proteins have a high sequence identity or share a high structural similarity. Based on the structural homologous, the β2-β3 hairpin of scyllatoxin, a scorpion toxin containing the CSαβ motif, is replaced by the CDR2-like loop of human CD4. The chimeric protein can bind to the HIV-1 envelope glycoprotein and the affinity is increased 100 fold as compared with the native scyllatoxin (Vita et al., 1999). In another case, VrD1 and VrD2, two defensins of *Vigna radiate*, share 80% sequence identities. The major difference of sequences concentrats on the loop 3. When the loop 3 of VrD1 is grafted to VrD2, the chimeric peptide exhibits the enzyme inhibitory

Combinatorial chemistry approach are powerful in screening and selecting binders with high affinity and high specificity (Hosse et al., 2006). It has been widely applied in antibody scaffold and thousands of antibodies are generated through the technology. Combinatorial chemistry approach also has been recruited to develop and isolate artificial proteins with new functions (Zhao et al., 2004). The approach could accelerate protein engineering based on a CSαβ motif to develop novel peptides with biomedical interesting (Thevissen et al., 2007;Van Gaal et al., 2004). Based on scaffold of insect defensin A, an expression library of peptides with 29 residues is constructed and used in screeening novel binders to targets. The expression library is artificially synthesized and amino acid of seven positions on the loops are randomized (Zhao et al., 2004). Tumor necrosis factor α (TNF-α), TNF receptor 1, TNF receptor 2 and antibody against BMP-2 are selected as targets and the screening results

Although several successful engineered peptides based on the CSαβ motif scaffold have been reported, to design new functions into the scaffold does perplex us. In this section, we would like to discuss challenges in engineering a CSαβ motif to equip desired functions.

To introduce tailored functions into a protein scaffold is never an easy task. For its ultra stability and high diversity of function, there is no doubt that peptide with a CSαβ motif is a suitable scaffold to be engineered for biomedical applications. To date, there are only a few of successful cases are reported. Many structures of peptides with CSαβ motif have been solved and deposited in database, but knowledge about the motif is poor. In spite of hundreds of peptides with a CSαβ motif deposited in the database, it is hard to make conclusions relating to structures and biochemical functions of the motif. Key residues are

but has the same circular dichroism spectrum profile as the native Chtx does.

**4.2 Functional epitope exchange** 

function as VrD1 does (Lin et al., 2007).

**4.3 Combinatorial chemistry approach** 

show significant enrichment in all cases.

**5.1 Lacking co-structural model** 

**5. The challenges of engineering of CS**αβ **motif** 

revealed in some peptides with a CSαβ motif and molecular docking models provide reasonable explainations (Figure 6). The real interaction is not clarified, for lacking of a costructural model. A co-structural model of the motif and its target will provide information about the dynamic interactions of the two protein molecules (Dumas et al., 2004;Thioulouse & Lobry, 1995). Structures of the complexes of peptides with a CSαβ motif and its counterparts will provide the situation of protein-protein interaction and have a great benefit to engineering of the scaffold.

(a) HIV reverse trascriptase with inhibitor (b) Docking model

Fig. 6. Docking model of plant defensin to HIV reverse transcriptase. Structures of HIV reverse transcriptase (3DRP, HIV RTase) and VrD2 (2GL1) are retrieved from the Protein data bank. Molecular docking is performed with PatchDock

(http://bioinfo3d.cs.tau.ac.il/PatchDock/). (a) The crystal structure of HIV RTase with inhibitor. (b) Molecular docking model of VrD2 and HIV RTase. Cyan: HIV RTase, yellow: VrD2 and red: HIV RTase inhibitor.
