Mayo K H, Haseman J, Ilyina E, Gray B
Department of Biochemistry, Biomedical Engineering Center, University of Minnesota Health Science Center, Minneapolis 55455, USA.
Biochim Biophys Acta. 1998 Sep 16;1425(1):81-92. doi: 10.1016/s0304-4165(98)00053-1.
Novel peptide 33mers have been designed by incorporating beta-conformation stabilizing residues from the beta-sheet domains of alpha-chemokines and functionally important residues from the beta-sheet domain of human neutrophil bactericidal protein (B/PI). B/PI is known for its ability to kill bacteria and to neutralize the action of bacterial endotoxin (lipopolysaccharide, LPS) which can induce septic shock leading to eventual death. Here, the goal was to make short linear peptides which demonstrate good beta-sheet folding and maintain bioactivity as in native B/PI. A library of 24 peptide 33mers (betapep-1 to betapep-24) were synthesized with various amino acid substitutions. CD and NMR data acquired in aqueous solution indicate that betapep peptides form beta-sheet structure to varying degrees and self-associate as dimers and tetramers like the alpha-chemokines. Bactericidal activity toward Gram-negative Pseudomonas aeruginosa was tested, and betapep-19 was found to be only about 5-fold less potent (62% kill at 1.2 x 10(-7) M) than native B/PI (80% kill at 2.9 x 10(-8) M). At LPS neutralization, betapep-2 and -23 were found to be most active (66-78% effective at 1.2 x 10(-6) M), being only about 50-100-fold less active than B/PI (50% at 1.5 x 10(-8) M). In terms of structure-activity relations, beta-sheet structural stability correlates with the capacity to neutralize LPS, but not with bactericidal activity. Although a net positive charge is necessary for activity, it is not sufficient for optimal activity. Hydrophobic residues tend to influence activities indirectly by affecting structural stability. Furthermore, results show that sequentially and spatially related residues from the beta-sheet domain of native B/PI can be designed into short linear peptides which show good beta-sheet folding and retain much of the native activity. This research contributes to the development of solutions to the problem of multiple drug-resistant, opportunistic microorganisms like P. aeruginosa and of agents effective at neutralizing bacterial endotoxin.
通过整合α-趋化因子β-折叠结构域中稳定β-构象的残基以及人中性粒细胞杀菌蛋白（B/PI）β-折叠结构域中功能重要的残基，设计出了新型33肽。B/PI以其杀灭细菌和中和细菌内毒素（脂多糖，LPS）作用的能力而闻名，LPS可诱导败血性休克并最终导致死亡。在此，目标是制备出具有良好β-折叠结构且能保持与天然B/PI相同生物活性的短线性肽。合成了一个包含24种33肽（betapep-1至betapep-24）的文库，这些肽具有不同的氨基酸取代。在水溶液中获得的圆二色光谱（CD）和核磁共振（NMR）数据表明，betapep肽能形成不同程度的β-折叠结构，并像α-趋化因子一样以二聚体和四聚体形式自缔合。测试了对革兰氏阴性铜绿假单胞菌的杀菌活性，发现betapep-19的效力仅比天然B/PI低约5倍（在1.2×10⁻⁷ M时杀菌率为62%）（天然B/PI在2.9×10⁻⁸ M时杀菌率为80%）。在LPS中和方面，发现betapep-2和-23最具活性（在1.2×10⁻⁶ M时有效率为66 - 78%），其活性仅比B/PI低约50 - 100倍（在1.5×10⁻⁸ M时为50%）。就构效关系而言，β-折叠结构稳定性与中和LPS的能力相关，但与杀菌活性无关。虽然净正电荷对活性是必需的，但对于最佳活性并不充分。疏水残基倾向于通过影响结构稳定性间接影响活性。此外，结果表明，天然B/PIβ-折叠结构域中顺序和空间相关的残基可以设计到短线性肽中，这些肽具有良好的β-折叠结构并保留了许多天然活性。这项研究有助于开发针对多重耐药性机会性微生物（如铜绿假单胞菌）问题的解决方案以及有效中和细菌内毒素的药物。
