Biol3176

1. Biol3176 Monday 27th August KcsA and Shaker Ion Channel Structures John Dedman LG1 9.00am Tuesday 28th August nAChR structure John Dedman LG1 10.00am

2. Dr Paul Carr Research School of Chemistry e-mail: pdc@rsc.anu.edu.au tel: x53766

3. Ion Channels • facinating membrane bound proteins • extensively studied physiologically and by mutagensis • resistant to structural analysis(only ~12 / 10,000 proteins in pdb from membranes)

4. Ion Channels - Properties • selective • 1:10000 between K+ and Na+featureless spheres 1.35 Å and 0.95 Å respectively. (1Å=10-10 m) • high throughput • 108 ions sec-1 (close to diffusion limit) • gated • rapidly opened and shut in response to voltage (eukaryotic) or pH change (bacterial)

5. Differences in tertiary structure of bacterial (KcsA) and voltage gated ion channels

6. Sequence alignment

7. Key Papers • Doyle et al. (1998) Science, 280, 69 - 77 • Clapham D.E. (1999) Nature Structural Biology, 6, 807 - 810 • Branden and Tooze (1998) Introduction to Protein Structure

8. X-ray crystallography in a nut shell Crystallisation P r o t e i n i n m e m b r a n e S o l u b i l i z e d p r o t e i n Calculate electron density maps Fit model and refine Collect X-ray diffraction Patterns

9. Crystallisation • 35 carboxyl terminal amino acids proteolytically removed • Detergent exchange dialysis with N,N-dimethyldodececylamine-N-oxide • sitting drop vapour diffusion with 48% PEG 400

10. Dielectrics and Dipoles • Dielectric: A non conductor of electric current. A substance in which an electric field gives no net flow of electric charge but only a diplacement of charge • Dipole: two equal point charges of opposite sign separated by a distance. The strength of a dipole is measured by its dipole moment = product of either charge and the distance between them

11. Coulomb’s Law F=force, q=charge D=dielectric constant D=1, vacuum/air r=distance, k=4π F q1 q2 k q1 q2 F= D r2 + H + + + H H O H - D=80 water q1 O q2 - + + H H O - oooooooooooooooooooooooo |||||||||||||||||||||||| q1 q2 D=2 lipid bilayer |||||||||||||||||||||||| oooooooooooooooooooooooo

12. Helical dipoles

13. Structure

14. Schematic

15. ion stabilisation

16. pore 12Å Selectivity Filter Wider ‘Lake’ lined by hydrophobics Acidic residues attract cations

17. Aromatics at interfaces

18. gating via inner helix rotation

19. Other accesory proteins associated the voltage gated ion channels

20. Shaker K • Key Paper: Three-Dimensional Structure of a Voltage-Gated Potassium Channel at 2.5nm Resolution Sokolova,O., Kolmakova-Partensky, L., and Grigorieff, N. Structure (March 2001), 9, 215-220

22. E l e c t r o n m i c r o s c o p y i n a n u t s h e l l S o l u t i o n o n P r o t e i n i n m e m b r a n e S o l u b i l i z e d p r o t e i n t h i n c a r b o n f i m l W a s h a n d s t a i n I m a g e w i t h p a r t i c l e s E l e c t r o n i n d i f f e r e n t p r o j e c t i o n s m i c r o s c o p y

25. 100 Å

26. Doyle et al. (1998). Science, 280, 69-77. Kreusch et al. (1998). Nature, 392, 945-48.