Transcript Lokey_guest_08

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
Scott and Smith, Science v. 249 (1990) p. 386
R1
R2
R3
R4
or
SS
R4
R3
R1
R2
HS
O
O
N
H
N
O
O
H
N
N
H
QuickTime™ and a Animation decompressor are needed to see this picture.
NH2
O
SH
O
H
N
O
N
HN
O
O
H2N
QuickTime™ and a Animation decompressor are needed to see this picture.
NH
S S
O
H
N
O
O
N
HN
O
O
NH
S S
H2N
O
O
H
N
O
N
HN
O
O
H2N
NH
S S
O
Fuzeon
QuickTime™ and a
TIFF (LZW) decompressor
are needed to see this picture.
Science v. 271 p. 1854 (1996)
Helical Wheels - to determine amphilphilicity
Native chemical ligation
--Steve Kent
Solid phase peptide synthesis
Cl
Cl
Cl
100-500 µ
Cl
500 nm
15 Å
HO
Cl
O
O
OH
HO
O
O
O
Cs2CO3
acid-labile linker
=
HO Linker
Fmoc Solid Phase Peptide Synthesis
H
N
O
O
Coupling Agent
OH
Fmoc
H
N
O
O
N N
N
O
N
Fmoc
O
H2N
O Linker
S
H
N
H2N
O
Base
O Linker
O
S
Fmoc
N
H
H
N
O
O Linker
O
S
O
N
H
HNFmoc
O
O
1. Chain elongation
(Fmoc peptide synthesis)
2. Allyl ester deprotection
O H X3 O H X5
X
N
N
N
N
NH2
2
4
O O
H X O H X O
NH
N
H
O
HO
1
O
H2N
1. Cyclization (Peptide coupling)
2. Cleavage (acid)
X1 H O X2
N
O
NH
O
HN
X3
NH
O
HN
O
N
4
X5 O H X
Gramicidin S - a cyclic peptide
antibiotic
Split pool synthesis
A
Linker
A
Linker
B
Linker
B
C
Linker
C
Pool
Linker
Linker
Linker
A
B
C
D
A
B
Linker
C
AD
Linker
BD
Linker
CD
E
Linker
Linker
Linker
A
B
C
Split
A
B
Linker
C
AE
Linker
BE
Linker
CE
F
A
B
C
Linker
AF
Linker
BF
Linker
CF
a
c
ti
v
it
y
Positional scanning
Linker
Linker
Linker
AXX BXX CXX
Linker
Linker
Linker
XDX XEX XFX
Linker
Linker
Linker
XXG XXH XXI
Most active predicted sequence: BDI
How to identify kinase
substrates?
O
HO
P
O
O
O
kinase
Signal output
Positional scanning library
Cantley et al. JBC v. 272 (1997) p. 952
OH
PKC isoforms
O
O
P
O
O
O
OH2
O
Fe
N
O
OH2
O
OH
O
N
O
e
F
O
O
O
O P
O
O
Wash, sequence
bound peptides
Results
Approaches to the study of protein function
1. Biochemistry
2. Genetics
3. 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
3a. Forward chemical genetics
Mayer et al., Science (1999) v. 286 pp. 971 - 974
control
+ monastrol
Target ID: the candidate approach
http://mc11.mcri.ac.uk/wro
ngtrousers.html
Kinesin movies
http://www.proweb.org/
kinesin/
Reverse Chemical
Genetics Kevan Shokat
Curr. Op. Chem. Biol
v. 5 p. 360 (2001)
Ellman, et al.