2. 1 Bi 1 “Drugs and the Brain” Lecture 18 Thursday, May 4, 2006 A.from mRNA to Protein B. Protein degradation Monday 5/8. Dr. Michael McIntosh will introduce psychiatric disease.

3. 2 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 from Lecture 3

4. 3 Protein synthesis and degradation A. synthesis B. degradation “proteolysis” Modified from Little Alberts Panel 2-5 protein + Greek, breakdown

5. 4 A. structure of transfer RNA (tRNA) base pairing stabilizes the structure 3 nucleotides Modified from Little Alberts Figure 7-23 4 3 = 64 possible codons; 61 encode one of the 20 natural amino acids; 3 “stop” codons

6. 5 the tRNA synthetase translates the genetic code, because it contacts (a) the amino acid (c) in some cases, other parts of the tRNA (b) the anticodon loop

7. 6 Two videos associated with protein synthesis Little Alberts CD-ROM Chapter 7 7.6, translation 7.7, polyribosome

8. 7 The discovery of the amber “stop”codon... a Caltech story from 1960 Harris Bernstein today Bob Edgar a petri dish “My help consisted of flaming a wire loop to sterilize it, then use it to repeatedly pick phage plaques from among hundreds on one petri dish and then inoculate two other petri dishes which had been seeded with host bacteria.... they were pessimistic about the outcome, and we agreed as sort of a joke that if the experiment did work out they would name mutants after my mother.” © Caltech

9. 8 When there is no tRNA for a codon, the ribosome falls off the mRNA; the protein stops. Bernstein’s discovery: the “amber” codon, one of three STOP codons “stop” anticodon (amber) CUA Mutant “amber suppressor” tRNA normal mRNA amber “stop” codon UAG another in-frame “stop” codon UAA, UGA normal mRNA UAG normal mRNA UAG Translation continues until the next in-frame UAA or UGA

10. 9 The ribosome is fairly stupid. We trick the ribosome into treating an amber ”stop” codon as a signal to incorporate an amino acid. The 3 ”stop” codons ordinarily have no tRNA (but Bernstein’s mutant had an amber tRNA) mutated mRNA amber “stop” codon UAG “stop” anticodon (amber) modified tRNA CUA Hijacking the genetic code: Unnatural amino-acid incorporation. protein measure

11. 10 The ribosome is fairly stupid. We trick the ribosome into treating an amber ”stop” codon as a signal to incorporate an amino acid. The 3 ”stop” codons ordinarily have no tRNA (but Bernstein’s mutant had an amber tRNA) mutated mRNA amber “stop” codon UAG “stop” anticodon (amber) modified tRNA CUA Hijacking the genetic code: Unnatural amino-acid incorporation. inject frog egg

12. 11 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 interface From lecture 3

13. 12 http://www.its.caltech.edu/~lester/Bi-1-2004/AChBP-2004-BindingSite.pdb The AChBP binding site “aromatic box” occupied by an acetylcholine analog (2004) http://www.its.caltech.edu/~lester/Bi-1/AChBP-2004-BindingSite.pdb cation-  interaction? From lecture 3

14. 13 Measured “dose-response” relations verify that an identified side chain governs agonist-receptor interactions wild type (tryptophan) phenylalanine http://www.axon.com/cs_Xpress_Animations.cfm The instrument (~ 90 MB!): From lecture 7

15. 14 Unnatural amino-acids define acetylcholine binding within 0.5 Angstroms Quantum-mechanical calculations of cation-  energy Electrophysiologically measured ACh binding energy Unnatural amino acid mutagenesis and electrophysiology agree with crystallography! From lecture 7

16. 15 B. Protein degradation is accomplished primarily by proteolytic enzymes The genome encodes hundreds of proteolytic enzymes. They vary in -- sequence specificity for the “cut” -- cellular expression -- organelle of expression

17. 16 The light chain of botulinum toxin is an proteolytic enzyme that cleaves synaptic vesicle fusion proteins from Lecture 9

18. 17 Cells often mark proteins for proteolysis by attaching strings of the protein, ubiquitin. modified from Little Alberts Fig 18-7 to be proteolyzed strings of ubiquitiin other protein

19. 18 modified from Little Alberts 1 st edition Fig 7-32 Controlled proteolysis takes place in the proteasome shorter

20. 19 Controlled, selective proteolysis is vital for healthy cells... Failed protein breakdown may help to cause some neurodegenerative diseases such as Huntington’s, Parkinson’s and Alzheimer’s Lectures 22, 26 below

21. 20 Who pays for all this research? The US National Institutes of Health http://maps.yahoo.com/maps_result?addr=&csz=Bethesda+MD&country=&new=1

22. 21 The US National Institutes of Health (NIH) Executive Branch Department of Health and Human Services Bethesda MD, Just outside Washington DC Across the street: Walter Reed Army Hospital Uniformed Services University of the Health Sciences Howard Hughes Medical Institute Some FDA Research 2006 NIH Budget $28.4 B 1. On-campus (“intramural”) research accounts for 10% 2. The majority of the budget is awarded as research grants (~ 9,500)

23. 22 http://www.nih.gov/icd/ National Institutes of Health: Institutes and Centers

24. 23 Bi 1 “Drugs and the Brain” End of Lecture 18 Monday 5/8. Dr. Michael McIntosh will introduce psychiatric disease.