WP4: NMR spectroscopy

Structure Elucidation

The chemical structures and elements of the relative stereochemistry will be determined on submilligram quantities of the cyclic peptides, using state-of-the-art nuclear magnetic resonance (NMR) spectroscopy with cryoprobes and sensitivity-optimized Shigemi tubes together with high-resolution, multistage mass spectrometry (MS). Substructures (peptide side chains and modified motifs) will be identified using 13C-HSQC, 13C-HMBC, 13C-H2BC, 1H,1H-DQCOSY/TOCSY and 1H,13C-HSQCTOCSY NMR, as well as ADEQUATE variants. Sequential assignment is achieved by 1H-13C long-range correlations and NOE hopping. The progress of pulse sequence development like pure shift- and weak coupling techniques will be constantly evaluated and implemented into the workflow.

Relative stereochemistry

Relative stereochemistry is determined from NMR data sensitive to 3D structure: 1H-1H and 1H,13C coupling constants (3-4JHH, 2-4JCH), through-space proximity data (1H,1H-NOE) and anisotropic data like residual dipolar couplings (RDCs) and residual chemical shift anisotropy (RSCA) in anisotropic-inducing media (stretch/compression gels and virus phage alignment). For flexible and chiral molecules is goes hand in hand with conformational analysis and optical methods/simulations.

Conformation analysis

The conformational space occupied by a molecule is sensitive to a number of NMR parameters, including NOEs, homo- and heteronuclear scalar couplings, RDCs and RSCAs. This data will be used together with Molecular Dynamics and DFT calculations to generate conformational ensembles accurately describing the average conformations in both aqueous solution and while interacting with membrane models.

Induced conformational changes upon membrane interactions reveal information about the mechanism of action as well as lipid bilayer permeation.

Membrane interactions

Nanodisc technology has proved to be robust concerning lipid composition and stable for months in room temperature.[1] The stability of the Nanodiscs together with the capacity to accommodate guest molecules will enable accurate measurements of a wealth quantifiable experimental parameters under full stoichiometric control of the number of discs and AMPs. This type of data has until now been unobtainable in solution due to the shortcomings of the traditional model systems.

Micelle               Nanodisc                    Bicelle                               Liposome

[1] Hagn, F.; Etzkorn, M.; Raschle, T.; Wagner, G. J Am Chem Soc 2013, 135, 1919–1925.

Molecules from WP1 and WP2 will be characterized inside specifically designed nanodisc membrane models. For this purpose, new methods will be designed to extract structural and dynamics parameters, induced disorder and membrane core hydration. Nanodiscs have so far not been widely applied in interaction studies between small AMPs and lipid bilayers. Our ambition is therefore to push the field further by attempting to extract structural data on the inter-molecular interactions between several peptides inside the bilayer, or on the membrane surface.

The membrane perturbing and penetrating effects will be probed inside different membrane models using new methods to extract structural- and dynamics parameters, induced disorder and membrane core hydration