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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.

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side chains

“peptide”or

amide bonds

link the

“backbone”or

“main chain”or

“-carbons”

Little Alberts Figure 2-22© Garland publishing

shortest: 9longest: 5500

20 types

from Lecture 3

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Protein synthesis and degradation

A. synthesis

B. degradation“proteolysis”

Modified from Little Alberts Panel 2-5

protein + Greek, breakdown

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A. structure of transfer RNA (tRNA)

base pairingstabilizes the structure

3 nucleotides

Modified from Little Alberts Figure 7-23

43 = 64 possible codons;

61 encode one of the 20 natural amino acids;

3 “stop” codons

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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

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Two videos associated with protein synthesis

Little Alberts CD-ROM Chapter 7

7.6, translation

7.7, polyribosome

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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

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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

H2N

CH

C

O

O

R

Mutant“amber suppressor”

tRNA

normal mRNA

amber “stop” codonUAG

another in-frame“stop” codonUAA, UGA

normal mRNA UAG normal mRNA UAG

Translation continues until the next

in-frame UAA or UGA

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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” codonUAG

“stop” anticodon(amber)

modified tRNA

CUA

H2N

CH

C

H2C

O

O

OO2N

Hijacking the genetic code: Unnatural amino-acid incorporation.

protein measure

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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” codonUAG

“stop” anticodon(amber)

modified tRNA

CUA

H2N

CH

C

H2C

O

O

OO2N

Hijacking the genetic code: Unnatural amino-acid incorporation.

inject

frog egg

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Binding region

Membrane region

Cytosolicregion

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 106

interface

From lecture 3

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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

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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

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Unnatural amino-acids define acetylcholine binding within 0.5 Angstroms

Quantum-mechanical calculations of cation- energyEle

ctro

phys

iolo

gica

lly m

easu

red

AC

h bi

ndin

g en

ergy

Unnatural amino acid mutagenesis and electrophysiology agree with crystallography!

From lecture 7

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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

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The light chain of botulinum toxin is an proteolytic enzyme that cleaves synaptic vesicle fusion proteins

from Lecture 9

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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

otherprotein

18modified from Little Alberts 1st edition Fig 7-32

Controlled proteolysis takes place in the proteasome

shorter

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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

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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

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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 HospitalUniformed Services University of the Health SciencesHoward Hughes Medical InstituteSome 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)

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http://www.nih.gov/icd/

National Institutes of Health:

Institutes and Centers

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Bi 1 “Drugs and the Brain”

End of Lecture 18

Monday 5/8.

Dr. Michael McIntosh will introduce

psychiatric disease.