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Bi 1 Lecture 3 Thursday, March 30, 2006 What is a Receptor? Receptors and Ion Channels as Examples of Proteins 1 receptor a molecule on the cell surface or in the cell interior that has an affinity for a specific molecule (the ligand). Latin, “to tie” Most drug receptors are proteins. Greek, “first” 2 shortest: 9 longest: 5500 “peptide” or amide bonds 20 types side chains link the “backbone” or “main chain” or “a-carbons” Little Alberts Figure 2-22 © Garland publishing 3 Proteins contain a few structural motifs: a helices (Swiss-prot viewer must be installed on your computer) http://www.its.caltech.edu/~leste r/Bi-1/alpha-helixalphabetical.pdb b sheets http://www.its.caltech.edu/~lest er/Bi-1/beta-sheetantiparallel.pdb Hide side chains Show H-bonds and distances Show ribbons & arrows Show side chains Show Van der Waals radii 4 Most drug receptors are membrane proteins Outside the cell nicotinic acetylcholine receptor nicotine, another agonist natural ligand (agonist) ~ 100 Å = 10 nm Membrane = lipid bilayer Inside the cell = cytosol (view in ~1995) 5 Overall topology of the nicotinic acetylcholine receptor (view in ~2000) 5 subunits each subunit has 4 a-helices in the membrane (20 membrane helices total) outside the cell: Binding Region Little Alberts figure 12-42 © Garland publishing 6 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) 5 subunits Little Alberts figure 12-42 © Garland publishing http://www.its.caltech.edu/~lester/Bi1/AChBP+Carb-5mer.pdb Color by chain Show 2 subunits, Chains, Ribbons 7 The AChBP binding site occupied by an acetylcholine analog (2004) http://www.its.caltech.edu/~lester/Bi-1/AChBP-2004-BindingSite.pdb http://www.its.caltech.edu/~lester/Bi-1-2004/AChBP-2004-BindingSite.pdb 8 Nearly Complete Nicotinic Acetylcholine Receptor (February, 2005) ~ 2200 amino acids in 5 chains (“subunits”), Binding region MW ~ 2.5 x 106 Membrane region Colored by secondary structure Colored by subunit (chain) Cytosolic region http://pdbbeta.rcsb.org/pdb/downloadFile.do?fileFormat=PDB&compression=NO&structureId=2BG9 9 How the binding of agonist (acetylcholine or nicotine) might open the channel: June 2003 view Ligand-binding region M1 M2 M3 M4 10 Most drug receptors are membrane proteins Some drugs compete with nicotine or acetylcholine Some drugs bind on the axis membrane region Nicotinic acetylcholine receptor 11 All I really need to know about life I learned in Bi 1 1. If you want a job done right, get a protein Protein Lecture # Ligand-gated Ion Channels 3 (today) Pumps and transporters 5, 13 Motors 10 G protein-coupled receptors and G proteins 12 Enzymes 13, 15 DNA-binding proteins 18 RNA polymerase, ribosome 18 Cystic Fibrosis Transmembrane Regulator 20 Rhodopsin 26 12 Protein structure prediction: An important 21st-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) 13 Protein Folding vs. “Inverse Folding” = Computational Protein Design Protein Folding (no degeneracy) Set of All Structures Inverse Folding (large degeneracy) Individual amino acids Set of All Sequences Several ways to make an arch 14 Bi 1 Cameo by Professor Pamela J. Bjorkman http://www.search.caltech.edu/CIT_People/action.lasso?-database=CIT_People&response=Detail_Person.html&-layout=all_fields&person_id=29067&-search X-ray Crystallography Crystal Growth X-ray Data Electron Density Protein Model 15 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 16 Overview of imaging No lens to refocus X-rays, so must understand reciprocal space and diffraction Diffraction: Scattering followed by interference 17 Bragg’s law Consider simultaneous reflection of a large number of x-rays. See diffraction maximum in direction q only if diffracted waves are in phase. Path difference (2dsinq) must represent an integral number of wavelengths to get constructive interference. 18 Learned two things from Bragg’s Law • sinq = nl/2 x 1/d Low angle: large interplanar spacing High angle: small interplanar spacing Since sinq 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 19 Same molecular transform sampled by different lattices a) Molecular transform b) Lattice c) Convolution of lattice and transform d - f ) Same molecular transform sampled by different lattices Modified from Lipson & Taylor, 1964 20 Resolution 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. From Harburn, Taylor, Wellbery, An Atlas of Optical Transforms 21 The electron density equation and the phase problem 1 (xyz) V h | F(hkl) | exp[( 2i(hk ky lz ) ia k Can measure this |F(hkl)|=I1/2 hkl ] l Can’t measure this • There are experimental methods for determining the phase for each reflection hkl. 22 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 23 Synchrotron x-ray sources e- • 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=hn=hc/l) • Is tunable!! Radiation emitted by accelerating charged particle tangent to path of circle 24