Afonin, S.

Brigaud, T.

Chaume, G.

Doan, V.

Grage, S. L.

Kara, S.

Ulrich, A. S.

English

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KIT – University of the State of Baden-Wuerttemberg and National Research Center of the Helmholtz Association Structure-function analysis of the  peptaibol Harzianin HK-VI Sezgin Kara1, Sergii Afonin1, Véronique Doan2, Stephan L. Grage1, Gregory Chaume2, Thierry Brigaud2, Anne S. Ulrich1,3 1 Institute of Biological Interfaces (IBG-2), Karlsruhe Institute of Technology; 2 Laboratoire SOSCO - Université de Cergy-Pontoise;  3 Institute of Organic Chemistry (IOC), Karlsruhe Institute of Technology Introduction  Peptaibols are naturally occurring peptides isolated from fungal sources. They contain a high amount of Aib (U) (α-aminoisobutyric acid), possess a C-terminal alcohol and N-terminal acetylation/alkylation. They are able to permeabilize bacterial membranes by pore formation. Harzianin HK-VI (HZ) is an short peptaibol (11-mer) isolated from Trichoderma pseudokoningii.[1]  1 2 3 ? Methods  Solid-state 19F-NMR and circular dichroism (CD) spectroscopy are used to determine the structure and alignment of HZ in lipid bilayers. For 19F-NMR analysis, we use α-trifluoromethylated (Tfm) amino acids (R)-, (S)-Tfm-Ala [2,3] and Tfm-Bpg [4] as substitutes of Aib and Leu/Ile residues, respectively.        Sequence, labelling strategy and the labels: (R)-Tfm-Ala (S)-Tfm-Ala Tfm-Bpg F3CNH2OHONH2OHOF3C CH3NH2OHOCH3 CF3Results References: [1] S. Rebuffat, S. Hlimi, Y. Prigent, C. Goulard, B. Bodo, J. Chem. Soc., Perkin Trans. 1 (1996) 2021; [2] F. Huguenot, T. Brigaud, J. Org. Chem. 71 (2006) 7075–7078; [3] D. Maisch, P. Wadhwani, S. Afonin, C. Böttcher, B. Koksch, A.S. Ulrich, J. Am. Chem. Soc. 131 (2009) 15596–15597; [4] P.K. Mikhailiuk, S. Afonin, A.N. Chernega, E.B. Rusanov, M. Platonov, G. Dubinina, M. Berditsch, A.S. Ulrich, I.V. Komarov, Angewandte Chem. 118 (2006), 5787-5789; [5] A.S. Ulrich, Progress in Nuclear Magnetic Resonance Spectroscopy 46 (2005) 1–21; [6] I. Segalas, Y. Prigent, D. Davoust, B. Bodo, S. Rebuffat, Biopolymers (1999) 71–85. Acknowledgements  We gratefully acknowledge the DFG/ANR for financial support. Special thanks to S. Roth, J. Bürck (CD12 beamline, ANKA) for their support with SR-CD/-OCD measurements. Hypothetical mechanisms how short pore-forming peptides can interact with lipid bilayers: by forming double-layered channels (1), by changing to an extended conformation (2), by causing membrane thinning (3). Peptide Sequence HZ   Ac-U-N-I-I-U-P-L-L-U-P-L-ol HZ 1-(R)-Tfm-Ala Ac-(R)-Tfm-Ala-N-I-I-U-P-L-L-U-P-L-ol HZ 1-(S)-Tfm-Ala Ac-(S)-Tfm-Ala-N-I-I-U-P-L-L-U-P-L-ol HZ 5-(R)-Tfm-Ala Ac-U-N-I-I-(R)-Tfm-Ala-P-L-L-U-P-L-ol HZ 5-(S)-Tfm-Ala Ac-U-N-I-I-(S)-Tfm-Ala-P-L-L-U-P-L-ol HZ 9-(R)-Tfm-Ala Ac-U-N-I-I-U-P-L-L-(R)-Tfm-Ala-P-L-ol HZ 9-(S)-Tfm-Ala Ac-U-N-I-I-U-P-L-L-(S)-Tfm-Ala-P-L-ol HZ 3-Tfm-Bpg Ac-U-N-Tfm-Bpg-I-U-P-L-L-U-P-L-ol HZ 4-Tfm-Bpg Ac-U-N-I-Tfm-Bpg-U-P-L-L-U-P-L-ol HZ 7-Tfm-Bpg Ac-U-N-I-I-U-P-Tfm-Bpg-L-U-P-L-ol HZ 8-Tfm-Bpg Ac-U-N-I-I-U-P-L-Tfm-Bpg-U-P-L-ol Representative MIC experiment. Strain: B. subtilis ATCC 6633. ALM: Alamethicin F30/3; GS: Gramicidin S; SC: solvent control; PC: positive control; NC: negative control. MIC [μg/ml] Strain HZ wt HZ Tfm-Ala peptides E. coli K12 > 256 > 256 S. aureus DSM 1104 128 > 256 S. xylosus DSM 20267 > 256 > 256 E. faecalis DSM 2570 > 256 > 256 B. subtilis ATCC 6633 > 256 > 256 MIC values for HZ. CD spectra of HZ peptides in H2O (A) and TFE (B); HZ wt (C) and HZ 5-(S)-Tfm-Ala (D) in DMPC vesicles (P/L 1/20). Deconvolution of the SR-CD spectrum for HZ wt in 50% TFE. -20000-15000-10000-500005000100001500020000180 200 220 240 260MRE [deg*cm2 /dmol] wavelength [nm] -15000-10000-50000500010000180 200 220 240 260MRE [deg*cm2 /dmol] wavelength [nm] HZ wtHZ 1R-TfmAlaHZ 1S-TfmAlaHZ 5R-TfmAlaHZ 5S-TfmAlaHZ 9R-TfmAlaHZ 9S-TfmAlaA C B D -15000-10000-5000050001000015000180 200 220 240 260MRE [deg*cm2 /dmol] wavelength [nm] 10°C25°C50°C-20000-15000-10000-500005000100001500020000180 200 220 240 260MRE [deg*cm2 /dmol] wavelength [nm] 10°C25°C50°Cα-helix               310-helix 16 Å 22 Å Ac--U N I I U P L L U P L -ol HZ wt ALM wt GS wt SC PC NC Peptide,  µg/ml 256 128 64 32 16 8 4 2 31P- and 19F-NMR spectra of Tfm-Ala-labeled HZ analogues in oriented DMPC lipid bilayers (P/L 1/100) as a function of temperature. CF3 dipolar splitting  values of Tfm-Ala labeled HZ peptides in DMPC and DPhPC lipid  bilayers (P/L 1/100). SR-OCD spectra: HZ wt in different lipid membranes at constant concentration (P/L 1/100) (A); HZ in DMPC at varying P/L (B). Putative alignment ( and  angles) and dynamics (Smol) of HZ in DMPC and DPhPC lipid bilayers assuming different models for secondary structure. Conclusions  HZ peptides   possess no antimicrobial activity  adopt a 310-helical structure when bound to the lipid membrane  show re-orientation in lipid membranes, dependent on the lipid composition and the lipid phase state Outlook   Synthesis of 15N-labeled HZ peptides and purification of Tfm-Bpg-labeled HZ analogues to obtain more NMR constraints  Investigation of the possibility of membrane thinning (2H-NMR, MD simulations)  Structure analysis of free HZ by NMR in solution and/or X-ray crystallography 5-(R) 5-(S) 9-(R) 40 20 0 δ 31P [ppm] 1-(R) 1-(S) 9-(S) Before 19F T=35°C TM 5-(S)-Tfm-Ala 9-(R)-Tfm-Ala 50 30 10 T [°C] isotrop. pos. 5-(R)-Tfm-Ala 50 30 10 T [°C] 9-(S)-Tfm-Ala 1-(S)-Tfm-Ala -50 -100 -150 δ 19F [ppm] 1-(R)-Tfm-Ala -50 -100 -150 δ 19F [ppm] -50 -100 -150 δ 19F [ppm] -50 -100 -150 δ 19F [ppm] -50 -100 -150 δ 19F [ppm] -50 -100 -150 δ 19F [ppm] TM Aims  We aim to solve the structure of membrane-bound HZ and understand its interactions with lipid bilayers.  N C 1    2  3 4  5   6   7  8   9  10 11   -10,0-5,0,05,010,015,020,025,0175 190 205 220 235 250 265OCD [mdeg] wavelength [nm] DMPCDPhPCDLPCDPPC-30,0-20,0-10,0,010,020,030,040,050,0175 190 205 220 235 250 265OCD [mdeg] wavelength [nm] P/L 1/200P/L 1/150P/L 1/100P/L 1/80P/L 1/40P/L 1/20A B Algorithm α 310 β turns PPII NRMSD CONTIN 31.3 10.4 0 14 9.8 0.253 SELCON3 29.8 10.2 -2.3 13.5 8.8 0.132 CDSSTR 39 10 5 11 9 0.005 Dipolar splitting [kHz] Lipid 1-(R) 1-(S) 5-(R) 5-(S) 9-(R) 9-(S) DMPC 0 4.9 7.4 -2.7 0 -4.0 DPhPC -1.2 5.2 4.8 0 4.6 -4.0 Structure model Lipid  [°]  [°] Smol RMSD 310-helix (ideal) DMPC 126 172 0.4 2.05 DPhPC 78 10 0.5 2.0 α-helix (ideal) DMPC 92 116 0.5 2.23 DPhPC 96 102 0.5 0.59 β-bend ribbon spiral [6] DMPC 102 118 0.6 1.38 DPhPC 124 106 0.4 0.87 Synthetic peptides are reconstituted in oriented lipid bilayers. From the 19F-NMR dipolar couplings, the structure and membrane alignment of peptides is determined.[5] Oriented CD with the synchrotron UV/VIS light source ANKA complements the results for the overall alignment of peptides.  310-helical conformation HPLC profiles of synthesized peptides: HZ wt crude (A) and pure (B), HZ 1-(S)-Tfm-Ala crude (C) and pure (D). A C D B contact: sezgin.kara@kit.edu 

Structure-funcion analysis of the peptaibol Harzianin HK-VI

https://publikationen.bibliothek.kit.edu/220098569/3816740

Structure-funcion analysis of the peptaibol Harzianin HK-VI

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