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Thèse Pour obtenir le grade de Docteur de l'Université Louis Pasteur de Strasbourg Discipline : Chimie Présentée par : Evgeniy S. SALNIKOV CNRS UMR 7177 Laboratoire de RMN et biophysique des membranes Etudes structurales de peptides antibiotiques de type peptaibol provoquant des modifications membranaires par spectroscopies RPE pulsée et RMN de l'état solide Soutenance prévue le 30-10-2007 devant la commission d'examen : Prof. Derek MARSH: Rapporteur externe Prof. Erick DUFOURC: Rapporteur interne Dr. Martial PIOTTO: Rapporteur interne Prof. Gerd KOTHE: Examinateur Dr. Jan RAAP: Examinateur Prof. Sergey A. DZUBA: Directeur de thèse Prof. Burkhard BECHINGER: Directeur de thèse

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cryo temperatures versus room temperature

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Publié par

profil-zyak-2012

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English

Poids de l'ouvrage

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Thèse

Pour obtenir le grade de Docteur de l'Université Louis Pasteur de Strasbourg Discipline : Chimie

Présentée par : Evgeniy S. SALNIKOV CNRS UMR 7177 Laboratoire de RMN et biophysique des membranes Etudes structurales de peptides antibiotiques de type peptaibol provoquant des modifications membranaires par spectroscopies RPE pulsée et RMN de l’état solideSoutenance prévue le 30102007 devant la commission d'examen : Prof. Derek MARSH: Rapporteur externe Prof. Erick DUFOURC: Rapporteur interne Dr. Martial PIOTTO: Rapporteur interne Prof. Gerd KOTHE: Examinateur Dr. Jan RAAP: Examinateur Prof.Directeur de thèseSergey A. DZUBA: Prof. Burkhard BECHINGER: Directeur de thèse

To my parents

Acknowledgements I gratefully thank my thesis directors, Prof. Sergey Andreevich Dzuba and Prof. Burkhard Bechinger for their endless optimism and wise supervision, which gives me an opportunity to progress as a scientist. I am indebted to Dr. Jan Raap, who introduces me into the wonderful world of membranemodifying peptides and actually made this thesis project possible. I also sincerely thank “SupraChem” project and his head Alexandre Varnek for the financial support during my stay in Strasbourg. I would like to extend my appreciation to my committee members: Prof. Derek Marsh, Prof. Gerd Kothe, Prof. Erick Dufourc and Dr. Martial Piotto for their useful comments and warm atmosphere during thesis defense. I’m especially grateful to Prof. Derek Marsh who agreed to revise the manuscript in such a short time period. I would like to express my gratitude to Dr. Philippe Bertani, who guided me in the field of solidstate NMR of oriented samples and always helped me during my stay in France. I thank Dr. Alexandre Milov for critical discussions of EPR experiments and Denis Erilov, James Mason, Jesus Raya, Roland Graff and Lionel Allouche who also participate in this work. I greatly appreciate the friendship of Svetlana Nedelkina and my interactions with Jesus Raya, Alexandre Milov and Leonid Kulik. I also thank the rest

members of both laboratories, the laboratory of NMR of condensed matter (Strasbourg, France) and the laboratory of chemistry and physics of free radicals (Novosibirsk, Russia), where I had luck to work. I would like to especially mention my brazilian friends Jarbas Magalhães Resende and Cléria Mendonça de Moraes for their cheerfulness. Finally, I am most grateful to my family who was always supporting me. I dedicate my doctoral thesis to my parents.

PhD thesis Evgeniy Salnikov

Contents AbstractAbbreviations 1. Introduction 2. Theory 2.1Structure and properties of lipid membranes 2.2Basic theory of Magnetic Resonance Relaxation EPR. Conventional Hahn echo pulse sequence NMR. Chemical shift 3. Materials and Methods 3.1Materials 3.2EPR of membranemodifying peptides: Preparation of MLVs samples ESEEM  access to peptide topology CW EPR spectra simulation  access to peptide olig omerization 3.3Solidstate NMR of oriented samples Preparation of oriented membrane samples 31 Control of the orientation of lipid bilayer by P NMRspectroscopy 15 Determination of the orientation of a helix by N NMRspectroscopy CrossPolarization NMR measurements PISEMA and peptide secondary structure Simulation of NMR experiments 3.4.Solidstate NMR of lipid vesicles Preparation of MLV samples2 H NMR of Labeled Phospholipids

13 1919 20 21

24 24 29

33 34 35 39 41 44

47 48

PhD thesis Evgeniy Salnikov

4. Location of Trichogin GA IV in lipid membrane by ESEEM Introduction 50 Results 52 Discussion 56 5. Alamethicin topology in a phospholipid membrane by Solid State NMR of oriented samples and EPR. Low and cryo temperatures versus room temperature: critical comparative study. Introduction 59 Results 61 Discussion 77 6. Structure and alignment of the membraneassociated peptaibols ampullosporin A 15 31 and alamethicin by oriented N and P solidstate NMR spectroscopy Introduction 81 Results 82 Discussion 97

7. Lipid membrane disturbance by the presence of alamethicin and ampullosporin A peptides. Perspective. Introduction 102 Results 103 Discussion 106 8. Summary108 15 Appendix. Preparation of N uniformly labelled ampullosporin A.112Bibliography.115Publications and Conference Abstracts 127

PhD thesis Evgeniy Salnikov

Abstract The structure, dynamics and topology of selected peptaibols when associated with biological membranes were characterized by magnetic resonance methods. Investigations were focused on the three members: trichogin GA IV, ampullosporin A and alamethicin, which are different in length and vary in their intensity of biological activity. 15 Using N uniformly labeled alamethicin and ampullosporin A, solidstate NMR of oriented samples allows to obtain information on the structure, dynamics and topology of peptides in their membrane bound state. Using TOAC (2,2,6,6tetramethylpiperidine1 oxyl4amino4carboxylic acid) labeled analogs of peptides alamethicin and trichogin GA IV, newly developed ESEEM (electron spin echo envelope modulation) approach allows to access peptide topology. In addition, CW (continuous wave) EPR of these TOAC labeled analogs gives information on peptide oligomerization. Studies suggest some common properties for the pept aibols when bound to membranes, suggesting similar mode of action. Namely: hydrophobic match / mismatch between peptide hydrophobic length and membrane apolar core was shown to play a key role on peptide orientation; a high 310 helical content in both alamethicin and ampullosporin A, when in a transmembrane state, was detected; and to the best of our knowledge, herein is the first time that oligomerization of transmembrane alamethicin molecules was directly observed. Another important aspect of this thesis work is the investigation of exactly the same peptaibol molecule, e.g. alamethicin, when bound to membranes using EPR and oriented solidstate NMR to obtain information on both the alignment and the oligomerization of the peptides in membranes. This approach using two complementary techniques allows not only the gathering of more information about peptide/membrane interactions but also the direct comparison of these different magnetic resonance methods.

PhD thesis Evgeniy Salnikov

Abbreviations:

Lipids:PC: phosphatidylcholine POPC: 1palmitoyl2oleoylsnglycero3phosphocholine DPPC: dipalmitoylphosphatidylcholine POPG: 1palmitoyl2oleoylsnglycero3[phosphorac(1glycerol)] POPE: 1palmitoyl2oleoylsnglycero3phosphoethanolamine POPS: 1palmitoyl2oleoylsnglycero3phosphoLserine DOPC: 1,2dioleoylsnglycero3phosphocholine Other: NMR: Nuclear Magnetic Resonance EPR: Electron Paramagnetic Resonance CW EPR: Continuous Wave EPR ESEEM: Electron Spin Echo Envelope Modulation MLV: multilamellar vesicle TOAC: 2,2,6,6tetramethylpiperidine1oxyl4amino4carboxylic acid PELDOR: pulsed electron–electron double resonance DQC: double quantum coherence CP: crosspolarization CD: Circular Dichroism Aib: AminoIsoButiryc acid Fmoc: 9Fluorenylmethoxycarbonyl HPLC: high performance liquid chromatography CSA: Chemical Shift Anisotropy CS: Chemical Shift

PhD thesis Evgeniy Salnikov

Chapter 1

Introduction

Peptaibols are linear antibiotic peptides of fungal origin, ranging in length from 5 to 20 residues. Their name derives from their chemical composition: “Pept” is the abbreviation of peptide, “Aib” indicates a high content of the unusual aminoacid Aib (aminoisobutiryc acid), and “ol” is due to the presence of a Cterminal 1,2amino alcohol; they also contain an Nterminal acetyl group. (Chugh et al., 2001).Presently this family of peptides encompasses more than 300 members (alam ethicins, suzukacilins, zervamicins, antiamoebins, ampullosporins, trichogins, etc), whose sequences are collected in the Peptaibol database (http://www.cryst.bbk.ac.uk/peptaibol) (Whitmore et al., 2003). Aib is a wellknown helixinducing amino acid, since the presence of the second methyl group on the C atom imposes strong stereochemical restrictions on the peptide backbone (Karle and Balaram, 1990; Pispisa et al., 2000a and 2000b), which exhibits a high tendency to adopt 310 orhelical conformations. Indeed all peptaibol structures determined so far are largely helical (Rebuffat et al., 1999). Peptaibols are potent antimicrobial agents, many of which appear to permeate biomembranes. Their activity potency is strongly biased towards the longest ones, e.g. alamethicin (for review, see e.g. Woolley and Wallace, 1992; Duclohier and Wroblewski, 2001). When added to lipid bilayers alamethicin exhibits a welldefined pattern of successive increases in conductance levels, each of a duration of a few milliseconds (Gordon and Haydon, 1972; Boheim, 1974) which resemble those seen in the presence of large voltage or ligandgated channel proteins (reviewed in (Woolley and Wallace, 1992; Sansom, 1993; Bechinger, 1997)). There is presently a consensus for the mode of action of the long peptaibols whose helical length matches the standard bilayer thickness with the “barrel stave” model. In addition, the understanding of their action could provide valuable information on membrane channel proteins, which are difficult to purify in quantitative amounts sufficient for structural studies. 5

PhD thesis Evgeniy Salnikov

The “barrel stave” model (Fig. 1.1) describes the f ormation of transmembrane pores/channels by bundles of amphipathic helices, which oligomerize like the staves of a barrel, so that their hydrophobic surfaces interact with the lipid core of the membrane and their hydrophilic surfaces point towards the interior of the pore, which is filled with water. Since this model requires peptide insertion into the hydrophobic core of the lipid bilayer, it is reasonable to assume that in this case peptide association with the target membrane is driven predominantly by hydrophobic interactions. The beststudied peptide for which this model seems to hold is alamethicin, which we will talk a lot in the present thesis.

Figure 1.1.The barrelstave model of antimicrobialpeptideinduced killing. In this model, the attached peptides oligomerize and insert into the membrane bilayer so that the hydrophobic peptide regions align with the lipid core region and the hydrophilic peptide regions form the interior region of the pore. Hydrophilic regions of the peptide are shown in red, hydrophobic regions of the peptide are shown in blue. (taken from Brogden, 2005)Peptaibols, especially those with helix length shorter than 18 residues, are not capable to completely span the lipid bilayer of normal thickness. Herein the situation is much less clear and several mechanisms of membrane activity have been proposed. Employing the parallel with cationic peptides, which also adopt surface orientation, the “carpetlike” and “toroidal pore” models have been proposed to explain the membrane disruption activity of short peptaibols.

PhD thesis Evgeniy Salnikov

In the “carpetlike” model (Fig. 1.2), peptides are in contact with the phospholipid head groups throughout the entire process of membrane permeation and do not penetrate into the lipid hydrophobic core neither do oligomerize, in contrast to the barrelstave mechanism. Peptides lay parallel to the membrane surface (with the hydrophobic face pointing towards the lipid core, and the hydrophilic face to water). Membrane permeation occurs only if there is a high local concentration of membranebound peptides (so that they form a “carpet”). In this case, the surface tension caused by peptide insertion in the headgroup region is released by the formation of transient membrane spanning pores, made up of dynamic peptidelipid supramolecular com plexes (Gazit et al., 1995; Matsuzaki, 2001). In these holes, the lipid bilayer bends back onto itself forming a toroidal structure. As a consequence, a fraction of peptide molecules translocates into the inner leaflet of the membrane, significantly reducing the peptide density in the outer layer, and leading to the closing of the pore. Furthermore, at higher concentrations, the peptide causes the disintegration of the membrane a nd the formation of micelles (micellization). In this mechanism, as the peptide interacts strongly with the phospholipid head groups, electrostatic interactions presumably play a crucial role.This model explains the activity of antimicrobial peptides such as ovispirin that orientate parallel (“inplane”) to the membrane surface (Yamaguchi et al., 2004).

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