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Peptides in chemistry and biology. Phage display Solid phase peptide synthesis and applications of synthetic libraries Native chemical ligation in protein synthesis. Scott and Smith, Science v. 249 (1990) p. 386. R 1. R 2. R 3. R 4. or. SS. R 1. R 4. R 2. R 3. Fuzeon. - PowerPoint PPT Presentation
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Peptides in chemistry and biology
1. Phage display
2. Solid phase peptide synthesis and applications of synthetic
libraries
3. Native chemical ligation in protein synthesis
R1
R3R2
R4
or
SSR1
R2R3
R4
Scott and Smith, Science v. 249 (1990) p. 386
NH
NNH
HN
O
O
O
NH2
OHS
SH
O QuickTime™ and aAnimation decompressorare needed to see this picture.
NH
N
HN
HN
O
O
O
H2N
O
SSO
QuickTime™ and aAnimation decompressorare needed to see this picture.
NH
N
HN
HN
O
O
O
H2N
O
SSO
NH
N
HN
HN
O
O
O
H2N
O
SSO
QuickTime™ and aTIFF (LZW) decompressor
are needed to see this picture.
Fuzeon
Science v. 271 p. 1854 (1996)
Helical Wheels - to determine amphilphilicity
Native chemical ligation--Steve Kent
100-500 µ 500 nm
Cl
Cl
Cl
Cl
15 Å
Solid phase peptide synthesis
ClOH
O
O
HO
Cs2CO3
O
OOHO
acid-labile linker
= LinkerHO
Fmoc Solid Phase Peptide Synthesis
HN
O
O
HN
O
OH
O
O N
NN
N
Coupling Agent
Fmoc
Fmoc
LinkerO
OH2N
S
LinkerO
OHN
S
O
NH
FmocBase
LinkerO
OHN
S
O
H2N
NH
HNO
OO
Fmoc
NH
NHO
OHO
ONH
X1 HN N
H
HN NH2
O
O
O
OX2
X3
X4
X5
1. Cyclization (Peptide coupling)2. Cleavage (acid)
H2NHN
O
O
OHN
X1
NH
NH
NH
HN
O
O
O
O
X2
X3
X4X5
1. Chain elongation (Fmoc peptide synthesis)
2. Allyl ester deprotection
Gramicidin S - a cyclic peptide antibiotic
Linker
A
B
C
A
Linker
B
Linker
C
Linker
Pool
A
Linker
B
Linker
C
Linker
Split pool synthesis
D
E
F
A
Linker
B
Linker
C
Linker
A
Linker
B
Linker
C
Linker
A
Linker
B
Linker
C
Linker
D D D
E E E
F F F
Split
A
Linker
B
Linker
C
Linker
A B C
A B C
A B C
AXX
Linker
BXX
Linker
CXX
Linker
XDX
Linker
XEX
Linker
XFX
Linker
XXG
Linker
XXH
Linker
XXI
Linker
Most active predicted sequence: BDI
Positional scanning
How to identify kinase substrates?
HO OPOOO
kinase
Signal output
Positional scanning library
Cantley et al. JBC v. 272 (1997) p. 952
O
POO
O
OH
PKC isoforms
OH
N O
FeO
O
O
OH2
OH2
N
O
O
O
OO
O P OO
Wash, sequencebound peptides
Results
Approaches to the study of protein function
1. Biochemistry2. Genetics3. Pharmacology
http://bio.research.ucsc.edu/people/kellogg/
Approaches to the study of protein function
1. Biochemistry - requires cell-free assay
2a. Forward genetics
mutagenize Screen for interesting phenotype
Trace phenoptype to mutation
2b. Reverse genetics
a. Directed deletions, point mutations
b. Gene silencing (siRNA)
c. Knock-out mice
Mayer et al., Science (1999) v. 286 pp. 971 - 974
3a. Forward chemical genetics
control
+ monastrol
http://mc11.mcri.ac.uk/wrongtrousers.html
Kinesin movies
http://www.proweb.org/kinesin/
Target ID: the candidate approach
Reverse Chemical Genetics Kevan ShokatCurr. Op. Chem. Biolv. 5 p. 360 (2001)
Ellman, et al.