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

• Antragstellerin: Professorin Dr. Anne Ulrich
• Fachliche Zuordnung: Biologische und Biomimetische Chemie
• Förderung: Förderung von 2012 bis 2016
• Projektkennung: Deutsche Forschungsgemeinschaft (DFG) - Projektnummer 208935089

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.

• DFG-Verfahren: Sachbeihilfen
• Internationaler Bezug: Frankreich
• Beteiligte Personen: Dr. Sergiy Afonin; Professor Dr. Thierry Brigaud