1. amyloid biomembranes crystalline proteins Sample preparation (etc) for MAS SSNMR of biomembranes David Middleton School of Biological Sciences

2. Reality What the world sees Sample preparation (etc) for MAS SSNMR

3. Associated factors (ligands, peptides, prosthetic groups) Protein purification and preparation of membranes angles static aligned bilayers magic angle spinning Solid-state NMR techniques for biomembranes SSNMR amorphous dispersion

4. MAS SSNMR of proteins in their natural membranes MAS SSNMR can be used to study membrane proteins purified from tissue or bacterial cell without removing them from the native membrane environment Planar or microsomal membranes isolated by centrifugation Proteins amenable to analysis are usually naturally abundant or can be overexpressed -Rhodopsin (95 % of total ROS disk membrane protein) -Nicotinic acetylcholine receptor -P-type ion pumps -porins -Transporters (bacterial) Advantage: straightforward non-perturbing preparation, stable and functional protein Disadvantage: Not amenable to labelling (eukaryotic), contaminants

5. Sample handling Native membranes remain fully hydrated and are sedimented by ultracentrifugation to produce a viscous gel. Gel is packed into an MAS rotor to as high a protein density as possible (aim typically for 10 nmoles or higher in a 50  l volume – i.e., 8-10 mg/ml for a 40 kDa protein). kidney membrane E. coli membrane 13 C CP MAS spectra of natural membranes show background signals from lipids and proteins. Spectra have same features regardless of the source of the membranes.

6. What information can be gained? The three dimensional structures of ligands (e.g., hormones and drugs) in their binding pockets can be determined by isotopically labelling the ligand but not the protein. Information about ligand structure can be gained without prior knowledge of the receptor structure (although it helps, of course). e.g., drug design

7. Intensity profile of uridine signal Nucleoside transporter NupC from E. coli Recent example: DQ excitation at rotational resonance uridine 1’ 6 4

8. Recent example: DQ excitation at rotational resonance

9. Mapping ligand binding sites 6 Å GalP

10. control pNPG How to confirm the selectivity of a ligand 13 C chemical shift (ppm) Switch expression on/off Displace with competitor ligand methylglucuronide

11. MAS SSNMR of reconstituted systems Membrane protein reconstitution involves removing the protein of interest from its native membrane and incorporating into a new, well-defined lipid bilayer. Advantages: eliminates contaminating proteins; can vary lipid composition systematically; study structure and function in isolation Disadvantages: requires much more work; may lose protein function altogether

12. Crude membranes with protein of interest Purification Detergent solubilisation Add lipids/remove detergent Detergent screen (BOG, DDM) Detergent concentration/CMC Solubilised protein function Affinity (His or FLAG tagged) MAS SSNMR of reconstituted systems Gel filtration Selective extraction Choice of lipids Dialysis (vesicles) Biobeads (planar) Functional characterisation

13. Purification Detergent solubilisation Add lipids/remove detergent MAS SSNMR of reconstituted systems Functional characterisation  -adrenergic receptor

14. Adapted from MacLennan and Kranias, 2003 Case study: regulation of cardiac calcium flux

15. sarcolipin (atrial and skeletal muscle) phospholamban (ventricular) The proteins of interest SERCA (skeletal and cardiac muscle)

16. Reconstitution of SERCA1 for NMR studies SERCA1 from rabbit skeletal muscle SR vesiclessolubilization Reconstitution of SERCA and regulatory protein SSNMR measurements

17. Reconstitution of SERCA1 for NMR studies free lipid mixed SERCA sucrose density gradient

18. Dynamics of the conserved C-terminus MGINTRELFLNFTIVLITVILMWLLVRSYQY Ring dynamics will be impaired if the rings interact with SERCA 13 C-labelled Tyr Is the conserved RSYQY sequence of sarcolipin important for interactions with SERCA?

19. Structural analysis of microcrystalline proteins GluR2 S1S2J (dimer) Ionotropic glutamate receptor 2 X-ray structure allosteric modulator

20. AB Structural analysis of crystalline GluR2 S1S2J Hanging dropSitting drop

21. B 0 and resolution 13 C CP-MAS spectra 400 MHz 800 MHz

22. B 0 and resolution 15 N CP-MAS spectra 400 MHz 800 MHz

23. Structural analysis of crystalline GluR2 S1S2J