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amyloid

Sample preparation (etc) for MAS SSNMR of biomembranes. David Middleton School of Biological Sciences. amyloid. biomembranes. crystalline proteins. Sample preparation (etc) for MAS SSNMR. What the world sees. Reality. Solid-state NMR techniques for biomembranes.

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amyloid

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

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

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

  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 ml volume – i.e., 8-10 mg/ml for a 40 kDa protein). E. coli membrane kidney membrane 13C 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. Recent example: DQ excitation at rotational resonance Nucleoside transporter NupC from E. coli uridine Intensity profile of uridine signal 1’ 6 4

  8. Recent example: DQ excitation at rotational resonance

  9. Mapping ligand binding sites GalP 6 Å

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

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

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

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

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

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

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

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

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

  20. A B Structural analysis of crystalline GluR2 S1S2J Hanging drop Sitting drop

  21. B0 and resolution 13C CP-MAS spectra 800 MHz 400 MHz

  22. B0 and resolution 15N CP-MAS spectra 800 MHz 400 MHz

  23. Structural analysis of crystalline GluR2 S1S2J

  24. Structural analysis of crystalline GluR2 S1S2J

  25. Structural analysis of crystalline GluR2 S1S2J

  26. Structural analysis of crystalline GluR2 S1S2J

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