Greathouse Denise, Vostrikov Vitaly, McClellan Nicole, Chipollini Juan, Lay Jack, Liyanage Rohana, Ladd Taylor
Department of Chemistry and Biochemistry, University of Arkansas, Fayetteville, Arkansas 72701, USA.
J Pept Sci. 2008 Oct;14(10):1103-10. doi: 10.1002/psc.1047.
Lactoferricin (LfB) is a 25-residue innate immunity peptide released by pepsin from the N-terminal region of bovine lactoferrin. A smaller amidated peptide, LfB6 (RRWQWR-NH2) retains antimicrobial activity and is thought to constitute the "antimicrobial active-site" (Tomita, Acta Paediatr Jpn. 1994; 36: 585-91). Here we report on N-acylation of 1-Me-Trp5-LfB6, Cn-RRWQ[1-Me-W]R-NH2, where Cn is an acyl chain having n = 0, 2, 4, 6 or 12 carbons. Tryptophan 5 (Trp5) was methylated to enhance membrane binding and to allow for selective deuteration at that position. Peptide/lipid interactions of Cn-RRWQ[1-Me-W]R-NH2 (deuterated 1-Me-Trp5 underlined), were monitored by solid state 31P NMR and 2H NMR. The samples consisted of macroscopically oriented bilayers of mixed neutral (dimyristoylphosphatidylcholine, DMPC) and anionic (dimyristoylphosphatidylglycerol, DMPG) lipids in a 3:1 ratio with Cn-RRWQ[&1-Me-W]R-NH2 peptides added at a 1:25 peptide to lipid ratio. 2H-NMR spectra reveal that the acylated peptides are well aligned in DMPC:DMPG bilayers. The 2H NMR quadrupolar splittings suggest that the 1-Me-Trp is located in a motionally restricted environment, indicating partial alignment at the membrane interface. 31P-NMR spectra reveal that the lipids are predominantly in a bilayer configuration, with little perturbation by the peptides. Methylation alone, in C0-RRWQ[1-Me-W]R-NH2, resulted in a 3-4 fold increase in antimicrobial activity against E. coli. N-acylation with a C12 fatty acid enhanced activity almost 90 fold.
乳铁蛋白素（LfB）是一种由胃蛋白酶从牛乳铁蛋白N端区域释放的含25个氨基酸残基的天然免疫肽。一种较小的酰胺化肽LfB6（RRWQWR-NH2）保留抗菌活性，被认为构成“抗菌活性位点”（富田，《日本儿科学报》。1994年；36：585-591）。在此，我们报告1-甲基色氨酸5-LfB6（Cn-RRWQ[1-Me-W]R-NH2）的N-酰化情况，其中Cn是具有n = 0、2、4、6或12个碳的酰基链。色氨酸5（Trp5）被甲基化以增强膜结合能力，并允许在该位置进行选择性氘代。通过固态31P NMR和2H NMR监测Cn-RRWQ[1-Me-W]R-NH2（下划线为氘代的1-甲基色氨酸5）的肽/脂质相互作用。样品由宏观取向的混合中性（二肉豆蔻酰磷脂酰胆碱，DMPC）和阴离子（二肉豆蔻酰磷脂酰甘油，DMPG）脂质双层组成，比例为3:1，并以1:25的肽与脂质比例添加Cn-RRWQ[&1-Me-W]R-NH2肽。2H-NMR光谱显示酰化肽在DMPC:DMPG双层中排列良好。2H NMR四极分裂表明1-甲基色氨酸位于运动受限的环境中，表明在膜界面处有部分排列。31P-NMR光谱显示脂质主要处于双层结构，肽对其干扰很小。在C0-RRWQ[1-Me-W]R-NH2中，仅甲基化就使对大肠杆菌的抗菌活性增加了3-4倍。用C12脂肪酸进行N-酰化使活性增强了近90倍。
