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MRes project presentation
- 1. “Environmental determinants of conformation and membrane activity in pH responsive peptides” MRes Molecular Biophysics King’s College London Giulia Armato Supervisor: Dr A. James Mason
- 2. Phospholipids and membranes •Physical filter •Structural support of the cell •Information exchange surface •Catalytic surface
- 3. Membrane composition varies among different types of cells Bacteria Negatively charged membrane Eukaryotic healthy cells Uncharged external surface Eukaryotic un-healthy cells Negatively charged external surface
- 4. Peptides possibly sensitive to pH Peptide Sequence βAmyloid DAEFRHDSGYEVHHQKLVFFAEDV(SG)NKGAIIGLMVG SG GVVIA LL-37 LLGDFFRKSKEKIGKEWKRIVQRIKDFLRNLVPRTES Charge +1 +6 N° aa MW 42 4513.8 Da 37 4532.4 Da
- 5. Amyloid and Alzheimer’s disease
- 6. Membrane asymmetry alteration can Membrane asymmetry alteration can alter Amyloid conformation alter Amyloid conformation And what role have divalent metal cations?
- 7. Antimicrobial peptides and their membrane activity models
- 9. •Different types of membrane composition can cause switch of conformation on these peptides ? • Aspartate and glutamate residues can influence peptides conformation in a pH-dependent way ? •Which roles divalent cations have on these peptides ?
- 10. βA [27-42] Amyloid SPPS: O’ Acyl iso peptide method βA [27-42] βA [27-42] βA [1-24] Reduced hydrophobicity
- 13. ESI spectrum of LL-37, single species 7x 6x 8x 5x 3x 4x
- 14. Circular Dichroism of LL-37 in solution pH dependent conformational changes Two pKa: •7.55 •4.90 2.63 9.27
- 15. Divalent cations have little effect on LL-37 in solution MgCl2 Two pKa: •8.21 •4.25
- 16. Liquid state NMR LL-37 pH titration pH 9.17 Glutamic acid ? pH 8.87 pH 8.54 pH 8.11 pKa = 4.45 pH 7.81 pH 7.39 pH 7.06 pH 6.80 pH 6.46 pH 6.07 pH 5.57 Arginine ? pH 5.10 pH 4.69 pH 3.96 pH 3.55 pH 3.06 pH 2.54 pKa = 8.84
- 17. LL-37 conformation within differently charged membrane surfaces along the pH range DMPC and LL-37 pKa 6.97 DMPC-DMPS and LL-37
- 18. Tryptophan fluorescence of LL-37 LL-37 in POPE-POPG vesicles LL-37 in solution
- 19. LL-37 divalent cations dependent - disorder effect of membranes POPE/POPG-d31 NiCl₂ MgCl ₂ CoCl₂ NaCl
- 20. LL-37 disordering effect is exclusively on anionic lipids only POPG/POPE-d31
- 21. Conclusions: • Surface charge of membranes can cause a switch of conformation • Glutamate and Aspartate residues are possibly implicated in LL-37 pH transitions • Divalent cations can modulate the interaction of LL-37 with membranes
- 22. Acknowledgments • • • • • • • Dr Andrew James Mason Dr Sukhi Bansal Dr Tam Bui Dr Andrew Atkinson Giacomo Evola Gianluca Bello Maria Chiara Arno
- 23. TOCSY (cyan) and NOESY (orange)
- 24. NOESY
Editor's Notes
- Proteins membrane are anchored to the cytoscheletrum and hence help on maintaining the cell shape Separates the intracellular components from the outer environment and therefore helps on maintaining the cytoplasm biochemistry intact. The movements of substances across the bilayer can be distinguished in passive or active, based on the request of energy. So small molecules such as ions can move across the membrane by osmosis or diffusion, while for bigger molecules like nutrients an active transport through membrane channel and transporter is required The presence of many enzymes on the lipid bilayer, that cover the production of intra-cellular messenger or regulates the lipids idrolysis. Also the AMP cyclic that breaks down ATP
- PC is normally sequestered to the inner membrane leaflet, but is exposed in the outer membrane leaflet of apoptotic cells. surface-exposed PS also serves as an “eat-me” signal for removal of apoptotic cells
- Aspartic and glutamic acids(in red) presents ionisable side chains (pka=4) 1-42 water-soluble isopeptide has a serine-glycine ester (in green), the solubility is increased and hence an easier isolation via HPLC Solid Phase Peptide Synthesis will be used in order to obtain those peptides, and hence being able to perform biophysical studies on them. Peptides possibly sensitive to pH and divalent cations
- Need to add something about different behaves within different environment
- In a highly organised micelle environment amyloid was present in an α-helical configuration, while amyloid was in a β-sheet configuration when surrounded by a less organised micelle environment. amyloid is seen to aggregate at a much faster rate in presence of lipid vesicles of different charges and structures, than in solution. In particular, charged lipid membranes induced a electrostatic driven surface accumulation of Aβ peptides, latterly toxic aggregates accelerated with a much higher rate than in a membrane-free environment
- Most cathelicidins are linear peptides with 23-37 amino acid residues, and fold into amphiphatic α-helix, or may also be small-sized molecules (12-18 residues) with beta-hairpin structures, stabilized by one or two disulphide bonds. Cathelicidins are coupled with the adaptive immune system response, in fact they are generally expressed in cells that are in direct contact with the external environment, such as the epithelial cells of intestine, airway or ocular surfaces. LL-37 has the capacity on transferring extracellular plasmid DNA into mammalian cells; it forms a complex with plasmid DNA released in the environment, and enter mammalian cells via endocytosis that involves lipid raft containing PG surface domain.
- After defining the key points of these 2 membrane active peptides, we set some characteristics that we wanted to clarify.
- 21000 to -15000
- 10000 to -10000
- There are many transitions such the two highlighted
- Confiming also that the membrane environment plays a crucial role on determine the activity of membrane active peptide.
- Hα region zoomed in