2. 1 Bi 1 Lecture 3 Thursday, March 30, 2006 What is a Receptor? Receptors and Ion Channels as Examples of Proteins

3. 2 receptor Most drug receptors are proteins. a molecule on the cell surface or in the cell interior that has an affinity for a specific molecule (the ligand). Latin, “to tie” Greek, “first”

4. 3 side chains “peptide” or amide bonds link the “backbone” or “main chain” or “  -carbons” Little Alberts Figure 2-22 © Garland publishing shortest: 9 longest: 5500 20 types

5. 4  helices  sheets http://www.its.caltech.edu/~leste r/Bi-1/alpha-helix- alphabetical.pdb http://www.its.caltech.edu/~lest er/Bi-1/beta-sheet- antiparallel.pdb Proteins contain a few structural motifs: Hide side chains Show H-bonds and distances Show ribbons & arrows Show side chains Show Van der Waals radii (Swiss-prot viewer must be installed on your computer)

6. 5 nicotinic acetylcholine receptor Most drug receptors are membrane proteins Outside the cell Inside the cell = cytosol (view in ~1995) natural ligand (agonist) nicotine, another agonist Membrane = lipid bilayer ~ 100 Å = 10 nm

7. 6 Overall topology of the nicotinic acetylcholine receptor (view in ~2000) outside the cell: 5 subunits each subunit has 4  -helices in the membrane (20 membrane helices total) Little Alberts figure 12-42 © Garland publishing Binding Region

8. 7 The acetylcholine binding protein (AChBP) from a snail, discovered in 2001, strongly resembles the binding region (Swiss-prot viewer must be installed on your computer) Color by chain Show 2 subunits, Chains, Ribbons 5 subunits Little Alberts figure 12-42 © Garland publishing http://www.its.caltech.edu/~lester/Bi- 1/AChBP+Carb-5mer.pdb

9. 8 http://www.its.caltech.edu/~lester/Bi-1-2004/AChBP-2004-BindingSite.pdb The AChBP binding site occupied by an acetylcholine analog (2004) http://www.its.caltech.edu/~lester/Bi-1/AChBP-2004-BindingSite.pdb

10. 9 Binding region Membrane region Cytosolic region Colored by secondary structure Colored by subunit (chain) Nearly Complete Nicotinic Acetylcholine Receptor (February, 2005) http://pdbbeta.rcsb.org/pdb/downloadFile.do?fileFormat=PDB&compression=NO&structureId=2BG9 ~ 2200 amino acids in 5 chains (“subunits”), MW ~ 2.5 x 10 6

11. 10 How the binding of agonist (acetylcholine or nicotine) might open the channel: June 2003 view M2 M1 M3 M4 Ligand-binding region

12. 11 Nicotinic acetylcholine receptor Most drug receptors are membrane proteins Some drugs bind on the axis Some drugs compete with nicotine or acetylcholine membrane region

13. 12 Protein Lecture # Ligand-gated Ion Channels 3 (today) Pumps and transporters5, 13 Motors10 G protein-coupled receptors and G proteins12 Enzymes13, 15 DNA-binding proteins18 RNA polymerase, ribosome18 Cystic Fibrosis Transmembrane Regulator20 Rhodopsin26 All I really need to know about life I learned in Bi 1 1. If you want a job done right, get a protein

14. 13 Protein structure prediction: An important 21 st -century problem Want to test your own skill at predicting protein structure? Then enter “Critical Assessment of Techniques for Structure Prediction” or CASP 7 http://predictioncenter.org/ Winners earn an automatic “A+” in Bi1 (retroactively, if appropriate)

15. 14 Protein Folding vs. “Inverse Folding” = Computational Protein Design Protein Folding (no degeneracy) Inverse Folding (large degeneracy) Set of All Structures Set of All Sequences Individual amino acids Several ways to make an arch

16. 15 X-ray Crystallography Crystal Growth X-ray DataElectron Density Protein Model http://www.search.caltech.edu/CIT_People/action.lasso?-database=CIT_People&- response=Detail_Person.html&-layout=all_fields&person_id=29067&-search Bi 1 Cameo by Professor Pamela J. Bjorkman

17. 16 X-ray crystallography Why X-rays? Right wavelength to resolve atoms Why crystals? Immobilize protein, enhance weak signal from scattering What is a protein crystal? Large solvent pathways (20-80% solvent) Same density as cytoplasm Enzymes active in crystals Are crystal structures valid compared with solution structures? Usually -- Compare NMR and X-ray structures Structures correlate with biological function Multiple crystal forms look same -- small effects of packing

18. 17 Overview of imaging No lens to refocus X-rays, so must understand reciprocal space and diffraction Diffraction: Scattering followed by interference

19. 18 Bragg’s law Consider simultaneous reflection of a large number of x-rays. See diffraction maximum in direction  only if diffracted waves are in phase. Path difference (2dsin  ) must represent an integral number of wavelengths to get constructive interference.

20. 19 Learned two things from Bragg’s Law sin  = n /2 x 1/d Low angle: large interplanar spacing High angle: small interplanar spacing Since sin   1/d, structures with large interplanar spacings (d) will have diffraction patterns with small spacings and vice-versa. Repeating unit in real space (crystal) --> diffraction maxima and minima

21. 20 Same molecular transform sampled by different lattices Modified from Lipson & Taylor, 1964 a) Molecular transform b) Lattice d - f ) Same molecular transform sampled by different lattices c) Convolution of lattice and transform

22. 21 Resolution From Harburn, Taylor, Wellbery, An Atlas of Optical Transforms An inverse Fourier transform (FT) including all of the high angle information gives back the original image. An inverse FT including only the low angle information gives back a low resolution view of Mickey.

23. 22 The electron density equation and the phase problem There are experimental methods for determining the phase for each reflection hkl. Can measure this |F(hkl)|=I 1/2 Can’t measure this

24. 23 X-ray detection Film (relic of the past) Diffractometers (almost relic of past, but used for small molecules) Multiwire detectors (almost relic of past) Phosphorimager detectors (R-AXIS, MAR) CCD detectors

25. 24 Synchrotron x-ray sources High-intensity x-ray emitted by charged particles accelerated in a curved path X-ray wavelength in range of 0.5 - 2 Å (from E=h =hc/ ) Is tunable!! Radiation emitted by accelerating charged particle tangent to path of circle e-e-