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Slide 1Slide 2Slide 3Slide 4Slide 5Slide 6Slide 7Slide 8Slide 9Slide 10Slide 11Slide 12Slide 13Unnatural amino-acids define acetylcholine binding within 0.5 AngstromsSlide 15Slide 16Slide 17Slide 18Slide 19Slide 20Slide 21Slide 22Slide 231Bi 1 “Drugs and the Brain” Lecture 18 Thursday, May 4, 2006A. from mRNA to Protein B. Protein degradation Monday 5/8. Dr. Michael McIntosh will introduce psychiatric disease.2 side chains“peptide”oramide bondslink the“backbone”or“main chain”or “-carbons”Little Alberts Figure 2-22© Garland publishingshortest: 9longest: 550020 typesfrom Lecture 33Protein synthesis and degradationA. synthesisB. degradation“proteolysis”Modified from Little Alberts Panel 2-5protein + Greek, breakdown4A. structure of transfer RNA (tRNA)base pairingstabilizes the structure3 nucleotidesModified from Little Alberts Figure 7-2343 = 64 possible codons;61 encode one of the 20 natural amino acids;3 “stop” codons5the 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 loop6Two videos associated with protein synthesisLittle Alberts CD-ROM Chapter 77.6, translation7.7, polyribosome7The discovery of the amber “stop”codon . . . a Caltech story from 1960Harris Bernstein todayBob Edgara 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.”© Caltech8When 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)CUAH2NCHCOORMutant“amber suppressor” tRNAnormal mRNAamber “stop” codonUAG another in-frame“stop” codonUAA, UGAnormal mRNAUAGnormal mRNAUAGTranslation continues until the next in-frame UAA or UGA9The 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 mRNAamber “stop” codonUAG“stop” anticodon(amber)modified tRNACUAH2NCHCH2COOOO2NHijacking the genetic code: Unnatural amino-acid incorporation.proteinmeasure10The 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 mRNAamber “stop” codonUAG“stop” anticodon(amber)modified tRNACUAH2NCHCH2COOOO2NHijacking the genetic code: Unnatural amino-acid incorporation.injectfrog egg11Binding regionMembrane regionCytosolicregionColored by secondary structureColored 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 106 interfaceFrom lecture 312http://www.its.caltech.edu/~lester/Bi-1-2004/AChBP-2004-BindingSite.pdbThe AChBP binding site “aromatic box” occupied by an acetylcholine analog (2004)http://www.its.caltech.edu/~lester/Bi-1/AChBP-2004-BindingSite.pdbcation- interaction?From lecture 313Measured “dose-response” relations verify that an identified side chain governs agonist-receptor interactionswild type (tryptophan)phenylalaninehttp://www.axon.com/cs_Xpress_Animations.cfmThe instrument (~ 90 MB!):From lecture 714Unnatural amino-acids define acetylcholine binding within 0.5 Angstroms Quantum-mechanical calculations of cation- energyElectrophysiologically measured ACh binding energyUnnatural amino acid mutagenesis and electrophysiology agree with crystallography! From lecture 715B. Protein degradation is accomplished primarily by proteolytic enzymesThe genome encodes hundreds of proteolytic enzymes. They vary in-- sequence specificity for the “cut”-- cellular expression-- organelle of expression16The light chain of botulinum toxin is an proteolytic enzyme that cleaves synaptic vesicle fusion proteins from Lecture 917Cells often mark proteins for proteolysis by attaching strings of the protein, ubiquitin. modified from Little Alberts Fig 18-7to be proteolyzedstrings of ubiquitiinotherprotein18modified from Little Alberts 1st edition Fig 7-32Controlled proteolysis takes place in the proteasomeshorter19Controlled, 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’sLectures 22, 26 below20Who pays for all this research?The US National Institutes of Healthhttp://maps.yahoo.com/maps_result?addr=&csz=Bethesda+MD&country=&new=121The US National Institutes of Health (NIH)Executive BranchDepartment of Health and Human ServicesBethesda MD, Just outside Washington DC Across the street: Walter Reed Army HospitalUniformed Services University of the Health SciencesHoward Hughes Medical InstituteSome FDA Research 2006 NIH Budget $28.4 B1. On-campus (“intramural”) research accounts for 10%2. The majority of the budget is awarded as research grants (~ 9,500)22http://www.nih.gov/icd/National Institutes of Health:Institutes and Centers23Bi 1 “Drugs and the Brain” End of Lecture 18 Monday 5/8. Dr. Michael McIntosh will introduce psychiatric


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CALTECH BI 1 - Drugs and the Brain

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