Tian et al. BMC Genomics 2010, 11:187 http://www.biomedcentral.com/1471-2164/11/187 Page 4 of 19 Figure 1 Multiple sequence alignment of navidefensins, nasonins and related peptides. 1: Amdefensin1(Am); 2: Amdefensin2(Am); 3: Navidefensin1-1(Nv); 4: Navidefensin1-2(Nv); 5: Navidefensin2-1(Nv); 6: Navidefensin2-2(Nv); 7: Navidefensin2-3(Nv); 8: Navidefensin3(Nv); 9: Phormicin(Pt); 10: Sapecin(Sp); 11: Dmdefensin(Dm); 12: Heliomicin(Hv); 13: Nasonin-1(Nv); 14: Nasonin-2(Nv); 15: Nasonin-3(Nv); 16: Nasonin-4 (Nv); 17: Nasonin-5(Nv); 18: Nasonin-6(Nv); 19: Nasonin-7(Nv); 20: Nasonin-8(Nv); 21: Nasonin-9(Nv); 22: Nasonin-10(Nv); 23: Nasonin-11(Nv); 24: Nasonin-12(Nv); 25: Nasonin-13(Nv); 26: Nasonin-14(Nv); 27: Cobatoxin-1(Cn); 28: Cll-Defensin1(Cll); 29: Ps-Termicin(Ps); 30: Dr-Termicin(Dr). Am: A. mellifera, Nv: N. vitripennis, Pt: Protophormia terraenovae, Sp: Sarcophaga peregrine, Dm: D. melonagaster, Hv: Heliothis virescens, Cn: Centruroides noxius, Cll: C. limpidus limpidus, Ps: Pseudacanthotermes spiniger, Dr: Drepanotermes rubriceps. Secondary structure elements (a-helix: cylinder; b-strand: arrow) and disulfide bridge connectivity are shown on the bottom of the alignment. Acidic residues in the propeptide are shown in pink and cleavage sites are boxed. Identical amino acids or constitutive replacements are shadowed in yellow and grey, respectively. Consensu/60% (Con.): - (negative), * (Ser/Thr), l (aliphatic), + (positive), t (tiny), a (aromatic), c (charged), s (small), p (polar), b (big), h (hydrophobic). Basic residues (K, R, H) are shown in blue. Residues split by phase 1 or 2 introns are boxed in green or purple. Omitted residues in carboxyl-termini are indicated by +aa. As mentioned previously, navitricins have two addi- tional cysteines which could form a fourth disulfide bridge. To confirm this, comparative modeling was again used to build the structure of navitricin-1 by choosing different templates, e.g. CITDs (sapecin and phormicin), the scorpion toxin BmTX1, and the plant sweet taste protein brazzein. In all these cases no acceptable models were generated and we thus tried to use the I-TASSER algorithm for structure simulation which produced a sui- table model, as evaluated by Verify 3D (Figure 3B). Over- all, this molecule is similar to the defensins described above including three identical disulfide bridges. Impor- tantly, this model correctly predicts the fourth disulfide bridge linking the n- and c-loops together which makes the whole structure more compact. In fact, computa- tional prediction was also applied to reveal an additional disulfide bridge in several insulin-like proteins [49]. (4) Biological activity Previous studies have highlighted a crucial role of a-helical and g-core regions of insect defensins in inhibiting the growth of Gram-positive bac- teria, whereas their n-loops could mediate antibacterial specificity [37,50,51]. Interestingly, all nasonins identified here have a shorter n-loop relative to CITDs. To confirm whether these novel DLPs have different antibacterial spectrum from CITDs, we chose nasonin-1 as a represen- tative. Reduced nasonin-1 was chemically synthesized and its in vitro refolded was performed by using reduced and oxidized glutathione (GSH/GSSG) under an alkaline condition (pH = 8.0). Refolded peptide was further puri- fied by reverse-phase high performance liquid chromato- graphy (RP-HPLC) (Figure 4A). In comparison with the reduced form, refolded nasonin-1 has a slightly earlier retention time (18 min vs. 19 min). The molecular weight (MW) of refolded nasonin-1 detected by matrix-assisted laser desorption/ionization time of flight mass spectrum (MALDI-TOF MS) is 3553.48 Da, about 7 Da smaller than the reduced form (3560.8 Da), in favor of the forma- tion of three disulfide bridges (Figure 4B).