Peptaibols as membrane-active antibiotics: Solid-state 19F-NMR structure-function analysis using tailor-made CF3-labeled amino acids

我们的目的是了解生物膜中的通道形成的分子机制的“肽”，这应该是通过组装成跨膜束透化细菌膜。由于这些抗微生物肽含有高比例的不寻常的α-氨基异丁酸（Aib），因此这些肽AIB醇难以合成和分析，因此它们的作用模式知之甚少。特别是一些非常短的序列不太可能跨越膜，但它们可以与典型的较长抗菌序列一样有效。为了深入了解它们的功能机制，设想使用固态19 F-NMR光谱对两种具有不同长度的膜包埋肽进行比较结构分析：众所周知的20-mer Alamethicin F30/3（ALM）和短的11-mer Harzianin HK-VI（HZ）。对于这些高灵敏度的NMR测量，需要设计特定的定制三氟甲基-（Tfm-）标记的氨基酸并将其掺入肽中。我们的目标是：-建立和优化对映体纯的α Tfm标记的氨基酸的合成，这些氨基酸被设计成适合Aib型肽骨架：（R-）和（S-）Tfm-丙氨酸（与Aib等排）; α-Tfm-脯氨酸和Tfm-假脯氨酸（脯氨酸的类似物，构成ALM和HZ中的特征性非螺旋Aib-Pro基序）; α-Tfm-谷氨酰胺和α-Tfm-谷氨酸（极性氨基酸的类似物，形成ALM中两亲性螺旋的极性面）。- 将这些α-Tfm-氨基酸掺入两种肽的不同位置，由于失活19 F-取代基的低反应性，这本身是具有挑战性的。- 分别使用定向圆二色谱和抗菌活性试验验证所有选择性标记肽类似物的构象和生物完整性。- 固态19 F-NMR分析脂膜中ALM和HZ的结构，以解析主链构象、分子排列、寡聚状态和动力学。- 在不同的脂质和相态的肽结构的比较，预计将提供洞察通道形成的关键步骤，即膜结合，膜插入，寡聚化，和重新定向。

Our aim is to understand the molecular mechanism of channel formation in biomembranes by “peptaibols”, which are supposed to permeabilize bacteria membranes by assembling into transmembrane bundles. As these antimicrobial peptides contain a high proportion of the unusual α-aminoisobutyric acid (Aib), such peptAIBols are difficult to synthesize and to analyze, hence their mode of action is poorly understood. Especially some very short sequences are unlikely to be able to span the membrane, yet they can be just as potent as the typical longer antimicrobial sequences. To gain insight into their functional mechanisms, a comparative structure analysis is envisaged, using solid-state 19F-NMR spectroscopy on two membrane-embedded peptaibols with different length: the well-known 20-mer Alamethicin F30/3 (ALM), and the short 11-mer Harzianin HK-VI (HZ). For these highly sensitive NMR measurements, specific tailor-made trifluoromethyl-(Tfm-)labeled amino acids need to be designed and incorporated into the peptides. Our goals are:- To establish and optimize the synthesis of enantiomerically pure αTfm-labeled amino acids that are designed to fit into the Aib-type peptide backbone: (R-) and (S-)Tfm-alanine (isosteric with Aib); α-Tfm-prolines and Tfm-pseudoprolines (analogues of proline, constituting characteristic non-helical Aib-Pro motifs in ALM and HZ); α-Tfm-glutamine and α-Tfm-glutamate (analogues of polar amino acids, forming polar face of amphiphilic helix in ALM). - Incorporation of these α-Tfm-amino acids into different positions of the two peptaibols, which is challenging per se due to the low reactivity of the deactivating 19F-substituents. - Verification of the conformational and biological intactness of all selectively labelled peptide analogues, using oriented circular dichroism and antimicrobial activity tests, respectively. - Solid-state 19F-NMR structure analysis of ALM and HZ in lipid membranes, to resolve the backbone conformation, molecular alignment, oligomerization state and dynamics. - Comparison of the peptide structures in different lipids and phase states is expected to give insights into the critical steps of channel formation, i.e. membrane binding, membrane insertion, oligomerization, and re-orientation.