**4.1 Site-direct mutagenesis**

638 Biomedical Science, Engineering and Technology

sodium ion currents from superior cervical ganglion neurons of rats (Dehesa-Davila et al., 1996;Possani et al., 1999). In the rat model, CII9 also exerts a depressant effect on the behavior and electroencephalographic activity and antagonizes the epileptiform activity induced by penicillin (Dehesa-Davila et al., 1996;Gazarian et al., 2005;Possani et al., 1999).

Peptides with a CSαβ motif act as primary defenders for their host. Microbe-killing abilities of plant defensins have been demonstrated and transgenic industrial crops have been bred to carry specific defensins to increase their tolerance to plant pathogens. For directly binding to membranes or cell walls, target pathogens do not easily resist peptides with a CSαβ motif as they do to protein-targeting antibiotics. Pathogenic microbes would escape attacking from conventional antibiotics by mutating proteins but it is never easy to change the properties of cell membranes or cell walls. Scaffold of a CSαβ motif could be as a suitable platform for developing peptide antibiotics. Some plant defensins are tested and show activities againt human pathogenic microbes. For example, Rs-AFP2, a defensin isolated from seeds of *Raphanus sativus*, has an inhibitory activity against *Candida albicans* (Landon et al., 2004;Terras et al., 1992;Thomma et al., 2003). Scientists of Novozymes, a Dnaish biotech company, isolated a novel plant defensin, plectasin, with excellent

Plectasin is a 40-amino acid residue peptide isolated from saprophytic ascomycete, *Pseudoplectania nigrella*, and might be the first plant defensin with a CSαβ motif isolated from fungus (Mygind et al., 2005). Plectasin expresses anti-bacterial activities against a broad Gram-positive bacteria and has an inhibitory effect at a low concentration of 0.25 μg/ml to growth of *Streptococcus pneumoniae* (Mygind et al., 2005). Plectasin also has a comparable killing rate to *Streptococcus pneumoniae* as do conventional antibiotics (Mygind et al., 2005). *Streptococcus pneumoniae* is a major pathogenic bacteria and the most common cause of hospital/community-acquired pneumonia, bacterial meningitis, bacteremia, sinusitis, septic arthritis, osteomyelitis, peritonitis, and endocarditis (Whitney et al., 2000). Currently, antibiotics are major treatments to patients with infection of *Streptococcus pneumoniae*. In recent years, *Streptococcus pneumoniae* is more and more resistant to antibiotics and the demand for new drugs to cure *Streptococcus pneumoniae* infection is urgent (Baquero et al., 1991;Whitney et al., 2000). Another challenge of theraping *Streptococcus* infection is that *Streptococcus* can be an intracellular pathogen and avoids targeting by the immune system or drugs (Gordon et al., 2000;Talbot et al., 1996). Different from the conventional antibiotics, plectasin can directly act on the cell wall precursor lipid II of *Streptococcus* (Mygind et al., 2005). Studies also showed that plectasin has an intracellular activity against *Streptococcus aureus* both in human monocytes and in mouse peritonitis model without effecting the cells viability or inducing IL-8 production

Based on the scaffold, some CSαβ proteins have been engineered to exhibit new functions or changing of antimicrobial activities (Lin et al., 2007;Vita et al., 1999;Vita et al., 1995). Different approaches, including minimal residue substitution, functional epitope exchange, structural based modification and combinatorial chemistry have been employed to engineer the scaffold to exhibit new functions (Lin et al., 2007;Thevissen et al., 2007;Van Gaal et al.,

**3.3 Peptide antibiotics: Plectasin** 

(Brinch et al., 2009;Hara et al., 2008).

**4. Protein engineering based on CS**αβ **motif scaffold** 

antibiotics ability.

In some studies, researchers focus on the relationships of structures, functions and each residue of peptides with a CSαβ motif. Extensive residue substitution is usually performed on the peptides and the changes of structures and functions are observed. The process is time-consuming and labor intensive but it is required for collecting basic information about the scaffold. Amino acid substitution has been extensively performed on two peptides with a CSαβ motif, VrD1 and brazzein (Assadi-Porter et al., 2010;Yang et al., 2009). In the two studies, molecular docking models are also established to investigate the interactions between receptors and ligands.

In both cases, similar positions along the structures are discovered that are crucial to functions of both peptides (Figure 5). These sites are widely distributed on β1 strand, loop 1, loop 2 and loop 3 (Assadi-Porter et al., 2000;Walters et al., 2009;Yang et al., 2009). From the molecular docking models, these sites either directly interact with their targets or play as functional epitopes and insert into the active site of their targets (Assadi-Porter et al., 2010;Liu et al., 2006;Yang et al., 2009). It is interesting that, when two negatively charged residues, D29 and E41, of brazzein are replaced by a positively charged amino acid, the sweetness of brazzein is greatly improved (Assadi-Porter et al., 2010). The mutants should be with high interests to industrial utilities. In the case of VrD1, there is no functional improvement observed on its mutants (Yang et al., 2009). Comparing conformations of the wild-type proteins and their mutations, there are only minimal shifts measured (Assadi-Porter et al., 2010). This implies that structure of peptide with a CSαβ motif is relatively stable and has high tolerance to amino acid substitution. Side chains of these residues on the interactive surface are crucial to biological functions of the peptides.

Fig. 5. Superposition of brazzein and VrD1. Some critical residues are overlapping between brazzein and VrD1. Red: brazzein, cyan: VrD1. Red and cyan labeled residues: critical residues of brazzein and VrD1; respectively. The two structures are aligned with SARST (http://sarst.life.nthu.edu.tw/iSARST/).

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