peptides in chemistry and biology

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.