Dr. Chiara CabreleEmmy-Noether Nachwuchsgruppe der DFG

(1)

F A K U L T Ä T F Ü R C H E M I E U N D P H A R M A Z IE U N IVERSITÄT REGENSBURG

Dr. Chiara Cabrele

E m m y-Noether Nachwuchsgruppe der DFG

Förderung: Deutsche Forschungsgemeinschaft (DFG) und Fonds der Chemischen Industrie (FCI)

(2)

Dr. C. Cabrele, E m m y-Noether Nachwuchsgruppe der DFG Fakultät für Chem ie und Pharmazie, Universität Regensburg

Forschungsthemen

Festphasensynthese von

Peptide, Proteine und Peptid m imetika

Aufklärung der

Sekundär- und Tertiär-Struktur von Polypeptiden Design von

Inhibitoren von Protein-Protein Wechselwirkungen

(3)

Peptide bestehen aus A m inosäuren, die durch eine A m idbindung verknüpfet sind

COOH R´

(Pg) HN_N H₂N CO(Pg)_C R´´

H₂N C R´

O

HN COOH

R´´

1. Kupplungsreagenzien

2. – (Pg)_N, – (Pg)_C

+

R

R BasischBasisch H istidin

H istidin (His, H) (His, H) Arginin

Arginin (Arg, R ) (Arg, R ) Lysin

Lysin ( (LysLys, K), K)

Sauer Sauer

Asparaginsäure

Asparaginsäure ( (AspAsp, D), D) G lutam insäure (

G lutam insäure (G luG lu, E), E)

Unpolar Unpolar

Phenylalanin

Phenylalanin ( (PhePhe, F), F) Alanin

Alanin (Ala, A ) (Ala, A ) Leucin

Leucin (Leu, L) (Leu, L) Isoleucin

Isoleucin (Ile, I) (Ile, I) Methionin

Methionin (Met, M ) (Met, M ) Prolin

Prolin (Pro, P) (Pro, P) Valin

Valin (Val, V) (Val, V) Tryptophan

Tryptophan ( (TrpTrp, W ), W ) Polar

Polar

Cystein

Cystein ( (CysCys, C), C) G lycin

G lycin ( (G lyG ly, G), G) G lutam in (

G lutam in (G lnG ln, Q), Q) Asparagin

Asparagin ( (AsnAsn, N), N) Serin

Serin (Ser (Ser, S), S) Threonin

Threonin ( (ThrThr, T), T) Tyrosin

Tyrosin (Tyr, Y) (Tyr, Y)

(4)

Die proteinogenen Aminosäuren

H₂N CH C CH₂

OH O

N

NH

H₂N CH C (CH₂)₃

OH O

NH C NH₂

NH

H₂N CH C (CH₂)₄

OH O

NH₂

H₂N CH C CH₂

OH O

SH

H₂N CH C H

OH O

H₂N CH C CH₂

OH O

CH₂ C NH₂

O

H₂N CH C CH₂

OH O

CH₂ C OH

O H₂N CH C

CH₂

OH O

C OH

O H₂N CH C

CH₃

OH O H₂N CH C

CH₂

OH O

H₂N CH C CH₂

OH O

CH₂ S CH₃ H₂N CH C

CH₂

OH O

CH CH₃ CH₃

H₂N CH C CH

OH O

CH₃ CH₂CH₃ H₂N CH C

CH₂

OH O

HN

C OH O

H₂N CH C CH

OH O

CH₃ CH₃

H₂N CH C CH₂

OH O

C NH₂

O

H₂N CH C CH₂

OH O

OH

H₂N CH C CH₂

OH O

OH H₂N CH C

CH

OH O

OH CH₃ H is (H )

Arg (R)

Lys (K)

Phe (F)

Leu (L)

Trp ( W )

A la (A )

M e t ( M )

Pro (P)

Cys (C)

Asn (N)

Thr (T)

G ly (G)

G ln (Q)

Ser (S)

Tyr (Y)

Ile (I) Val (V )

Asp (D)

G lu (E)

(5)

Polymerträger

Peptidketten gebunden am Polymerträger

Synthese

Linker

Linker Linker

Kupplungs- reagenzien

H G P D

E

L

Linker

Linker Linker

(6)

Festphasensynthese

Ankergruppe: Chlorid, Alkohol

L i n k e r

L i n k e r L i n k e r

L i n k e r

Cl

Cl NH(Pg)CH(R)COOH

Base

Cl Cl

OC(O)CH(R)NH(Pg) H[NHCH(R)CO]_nOH 1. Synthese

2. Abspaltung mit 1 % TFA

OH

O NH(Pg)CH(R)COOH

Aktivierungsreagens, Base H[NHCH(R)CO]_nOH

1. Synthese

2. Abspaltung mit

> 80 % TFA

OH O

4-Benzyloxybenzyl Alkohol

2-Chlortrityl Chlorid

NH O

O

OMe OH

NH(Pg)CH(R)COOH

Aktivierungsreagens, Base H[NHCH(R)CO]_nOH

1. Synthese 2. Abspaltung mit

1-5 % TFA

NH O

O

OMe OH

4-Hydroxymethyl-3- Methoxyphenoxybutter- säure Benzhydrylamin

OC(O)CH(R)NH(Pg)

(7)

Festphasensynthese Ankergruppe: A m in

L i n k e r

H[NHCH(R)CO]_nNH₂ 1. Synthese

2. Abspaltung mit 1 % TFA

A m ino-xanthen-3- yloxy

NH(Pg)CH(R)COOH

Aktivierungsreagens, Base

1. Synthese

2. Abspaltung mit

> 5 % TFA 4-(2´,4´-Dimethoxyphenyl-

aminomethyl-phenoxy NH₂

O O

NH(Pg)CH(R)COOH

Aktivierungsreagens, Base

NH₂

O O

COCH(R)NH(Pg)

H[NHCH(R)CO]_nNH₂

NH₂

O

OMe

NH

O

OMe

COCH(R)NH(Pg)

(8)

Festphasensynthese Ankergruppe: A ldehyd

L i n k e r

4-Formyl-3- methoxyphenoxy

O

OMe

H O

O

OMe

HN R RNH₂/TMOF

DCM/NaBH(OAc)₃

O

OMe

RN

R´

O

OMe

RN

S

O O

R´

R´COOH/HOAt/DIC

R´SO₂Cl/TEA

25 % TFA 5 % TFA

R´ NHR O

S

O O

R´ NHR

(9)

Festphasensynthese Ankergruppe: Sulfamyl

L i n k e r

4-Sulfamyl-butyryl

HN RCOOH/DIC/MeIm

ICH₂CN/DIPEA

R´NH₂

R NHR´

O O

(CH₂)₃ S

O O

NH₂ H

N

O

(CH₂)₃ S

O O

HN

O R

HN

O

(CH₂)₃ S

O O

N

O R CN

R OR´

O

R OH

O R SR´

R´OH OH ^- O

R´SH

(10)

Festphasensynthese Aufbau der Peptidkette

Lin k e r

Lin k e r Lin k e r

Lin k e r

Lin k e r Lin k e r

Lin k e r

Lin k e r Lin k e r

Linker CO

HN

Orthogonale Strategie:

Linker

Base-labil

Base-stabil

Säure-labil

Säure-stabil

Fmoc/t-Butyl Boc/Benzyl

(11)

Festphasensynthese Fmoc-Chemie

Linker CO

HN

Lin k e r CO HN CO Lin k e rLin k e r HN

CO HN

CH₃ C H₃C

CH₃ Ph

C Ph

Ph

Ph C Ph

Ph CH₃

C H₃C

CH₃

O C O

O O

t-But Trt

Trt Boc

Fluorenylmethyloxycarbonyl (Fmoc)

> 50 % TFA

Piperidin

Abspaltung vom H arz

(12)

Festphasensynthese Boc-Chemie

Linker CO

HN

Lin k e r CO HN CO Lin k e rLin k e r HN

CO HN

Bzl Bzl

H₂, Pd/C

20-50 % T F A Boc

H F

Abspaltung vom H arz

(13)

Aufbau der Peptidkette: Verknüpfung der A m inosäuren

Linker CO

H₂N COOH

HN

Lin k e r

Lin k e r Lin k e r

Lin k e r

Lin k e r Lin k e r

Lin k e r

Lin k e r Lin k e r

Aktivierung

COA HN

Linker CO

HN CO HN

(14)

COOH HN

Lin k e r

Lin k e r Lin k e r

Lin k e r

Lin k e r Lin k e r

Lin k e r

Lin k e r Lin k e r

Aktivierung

R O

N₃

R O

O O

R´

R O

F

R O

O N

N N

R O

O F

F

F F

Azid

Anhydrid

Fluorid (Chlorid)

Benzotriazolester

Pentafluorphenylester

(15)

COOH HN

Lin k e r

Lin k e r Lin k e r

Lin k e r

Lin k e r Lin k e r

Lin k e r

Lin k e r Lin k e r

N N N

O

Me₂N NMe₂

+

DIPEA

R O

O N

N N

HBTU

PF₆^-

N C N

DIC

R O

O N

NH

OH N N N

HOBt

C O

NH N H

(16)

Reinigung und Charakterisierung von Peptiden und Proteindomänen

Flüssigkeitschromatographie (präparative und analytische H P L C ) Massenspektrometrie (ESI, M A L D I-TOF)

A m inosäuresenquenzierung/Aminosäure-Analyse Kontrolle der Racem isierung der A m inosäuren

C irculardichroismus N M R

Fluoreszenz

Röntgen-Struktur

(17)

Reinigung von Peptiden: HPLC

H₂O /TFA

C H₃CN/TFA

210-220 nm (Peptidbindung)

274-280 nm (Tyr, Trp)

Si Si Si Si Si

OH OSi OH OSi OSi

n

pH t_R

2 7 12

H A

A^- B H⁺

B

(18)

Peptidreinigung: HPLC

min

5 10 15 20 25 30 35 40

mAU

0 50 100 150 200

MWD1 A, Sig=220,4 Ref=off (Q:\HPLC\CABRELE\LC920_19.D)

1.719 1.945 2.280 14.285 22.786 28.988 29.526 29.748 43.393 44.254

Ac-(66-106)-hID 1

Säule: C18

Detektion: 220 nm Fluss: 1 m l/m in

Gradient: 10-80 % B in 40 m in

(19)

Circulardichroismus

optisch optisch aktives aktives Substanz Substanz

n_R = n_L _εε_R = εε_L

Absorption Brechungsindex

L_R L_L

αα

(20)

(21)

190 200 210 220 230 240 250 260

-20 -10 0 10 20 30

190 200 210 220 230 240 250 260

-20 -15 -10 -5 0 5 10 15 20 25 30 35

190 200 210 220 230 240 250 260

-20 -10 0 10 20 30

190 200 210 220 230 240 250 260

-20 -10 0 10 20 30

190 200 210 220 230 240 250 260

-20 -10 0 10 20 30

190 200 210 220 230 240 250 260

-20 -15 -10 -5 0 5 10 15 20 25 30 35

190 200 210 220 230 240 250 260

-20 -10 0 10 20 30

c=0.37 mM/L c=0.288 mM/L

c=0.216 mM/L

c=0.103 mM/L

λ (nm)

c=0.154 mM/L

c=0.03 mM/L

[Θ] R x 10-3 (deg cm2 mol-1 )

c=0.247 mM/L

H1H1 H2H2

LoopLoop

N N CC

Ac-(66-106) hID1 Ac-(66-106) hID1

(22)

Precursor

Precursor CellCell

Differentiated

Differentiated CellCell DNADNA

Transcription Transcription

Factor

Factor (TF) (TF)

DNA-TF

DNA-TF ComplexComplex

T

Inhibition of DNA Binding Inhibition of DNA Binding

T

Inhibition of

Inhibition of Cell DifferentiationCell Differentiation

(23)

Subfamily of the Superfamily of the

Helix-Loop-Helix (HLH) Transcription Factors

mammals

H1H1 H2H2

LoopLoop

NN CC

ID1 ID2 ID3 ID4

EMC

Drosophila

Robert Benezra

Cell Biology Program,

Memorial Sloan-Kettering Cancer Center New York, NY, USA

1990: Identification of the ID Proteins

(24)

ID1 ID2 ID3 ID4 EMC

155 134 119 161 199

66-106 36-76 41-81 65-105

36-76

Subcellular location: nucleus

Subunit: heterodimer with other HLH proteins. ID3 can build also homodimers

neuro-/angiogenesis; absent in adult brain

early fetal tissue, including CNS; present in adult cerebellum lung; kidney; adrenal gland; absent in adult brain

neuronal maturation of CNS and PNS

wing morphogenesis; sensory organ development

Protein HLH

domain Length

(AA)

Expression

(25)

cell fate determination

embryogenesis/organogenesis

cell growth

cell cycle progression

apoptosis

angiogenesis in vivo

cell differentiation

tumor invasiveness

(26)

Action: Dominant negative regulators (inhibitors) of basic- HLH (bHLH) transcription factors

Mechanism: Segregation of the bHLH transcription factors by formation of inactive heterodimers

Lock and key principle

Ascorbate peroxidase- ascorbate-sodium Nat. Struct. Biol. 10, 303 (2003)

Protein domain-domain interaction

c-Myc-Max heterodimeric LZ J. Mol. Biol. 281, 165 (1998)

(27)

Regulators of the DNA-transcription (>200 identified thus far) by repressing or activating specific genes;

Key players of gene expression in cell cycle, cell proliferation and differentiation, development of haematopoietic, myogenic, pancreatic and neurogenic cell lineages, sex determination;

Classified in four main groups on the basis of the presence/absence of functional domains along with HLH:

bHLH-b PASPAS bHLHb

ID

HLH

bHLH-b LZLZ

b = b = basic basic regionregion;; LZ = LZ = leucineleucine zipperzipper

(28)

H₂N CH C

CH2

OH O

CH CH3 CH3

Leu

LZ HLH

H1H1 H2H2

LoopLoop

i i+4 i+8

(29)

(30)

HLH motif: essential for protein-protein interaction (homo-/hetero- dimerization)

Basic

Basic region:region: essential for DNA-binding (interaction with the core sequence CANNTG, E-box)

HLH:

HLH: about 50 about 50 residues, residues, twotwo amphipathic

amphipathic αααα-helices-helices

b: b: mostly mostly basicbasic residuesresidues

Lineage specific gene TranscriptionalTranscriptional activation activation

H1H1 H2H2 LoopLoop

NN CC

H1´H1´

H2´H2´

Loop´Loop´

N´N´

C´C´

bb

(31)

Classification on the basis of the tissue distribution, dimerization capabilities, DNA-binding specificities

Class I/Group A

E-proteins: E12, E47, HEB, E2-2 ubiquitous

homo-/heterodimers

Class II/Group A

MyoD, myogenin, Myf5/6, atonal, neuroD, ac-s tissue specific

heterodimers with Class I

Class III/Group B Myc, TFE, SREBP, Mi

LZ: yes

Class V/Group D ID1-4, EMC

heterodimers with Class I and II Class VI/Group B

Hairy

Proline in the basic region Class IV/Group B

Mad, Max, Mxi

homo- or heterodimers with Myc LZ: yes

Class VII/Group B/C Arnt, SIM

PAS domain

(32)

HLH motif: protein-protein interaction Basic

Basic regionregion: : absent in ID

Lineage specific gene

NoNo

transcriptional transcriptional

activation activation

H1 H1

H2H2 LoopLoop

NN

CC

H1´H1´

H2´H2´

Loop´Loop´

N´N´

C´C´

b´b´

(33)

helix 1

hID1 (52-108)

KVEILQHVIDYIRDLQLE

hID2 (22-78)

KMEILQHVIDYILDLQIA

hID3 (26-83)

QVEILQRVIDYILDLQVV

Max (22-77)

ADKRAHHNALERKRRDHIKDSFHSLRDSVP RAQILDKATEYIQYMRRK

basic region loop helix 2

helix 1

E47

(335-392) basic region loop helix 2

RLPALLDEQQVNVLLYDMNGCYSRLKELVP

ISRSKTPVDDPMSLLYNMNDCYSKLKELVP

GRGKGPAAEEPLSLLDDMNHCYSRLRELVP

TLPQNRKVS

SIPQNKKVS

GVPRGTQLS

SLQGEKAS

KSDKAQT

helix 1

MyoD

(108-163) basic region loop helix 2

NQRLP

KLLILQQAVQVILGLEQQ

KVEILRNAIRYIEGLQAL 1

1 1010 2020 3030 4040 5050

RERRMANNARERVRVRDINEAFRELGRMCQMHL

ADRRKAATMRERRRLSKVNEAFETLKRCTSSNP

(34)

Pho4-DNA

H1

H2 H2´

Max-DNA

H1

H2 H2´

H1´

LZ´

LZ

MyoD-DNA

H1

H2 H2´

H1´

Homodimers with a parallel, left-handed four-helix bundle

V50 Y70

P51 R60

I39 F43 L64´I63

L46 A67 I71 I71´

Y70´

M74 M74´

Residues of the hydrophobic core

(35)

When and Where are ID present?

During development, in many organs and tissues and in the nervous system;

ID1,2,3 show overlapping expression patterns, while ID4 has a unique pattern during embryogenesis;

ID2,4 are expressed also in specific neurons in the adult brain and cerebellum.

no apparent phenotype in the CNS

small olfactory bulb, slightly narrow frontal cortex, reduced number of NK cells, lack of lymph nodes, immature mammary glands (lactation defect), decrease in mature sperm

death in utero, small brain (premature withdrawal of neuroblasts from the cell cycle), defects in

angiogenesis

severe malformations, small and/or disorganized thalamus and

hypothalamus, reduced number of oligodendrocytes in these brain regions

Knock-out mice

ID1 ID3

ID2

ID1 ID3

ID4

(36)

... Cell Differentiation

a. One-way type

bHLH ID

precursor differentiated cell

(e.g. neuronal differentiation)

b. Bifurcation type

ID bHLH precursor

differentiated cell Y differentiated cell X

(e.g. NK cells development)

... Proliferation

ID

immature cell immature cells

... Survival

ID

differentiated cell differentiated cell

(37)

ID2 Rb, bHLH, Ets, Pax

anaplasia proliferation

ID1,3 metalloproteinases integrins, MMP2

angiogenesis tissue invasion

VEGF signaling

Lineage-specific bHLH

(38)

glioblastoma, medulloblastoma ID1,3

pancreatic cancer

ID1,2,3

seminoma

ID1,2,3,4

thyroid ID1

breast cancer ID1

ID1

cervical and endometrial cancer neuroblastoma ID2

neuroblastoma

ID2

melanoma

ID1,2,3 ID1

colorectal cancer

(39)

ID2 binds to the tumor suppressor retinoblastoma (Rb) protein.

Abnormal production of ID inactivates the Rb pathway directly (ID2) and indirectly through inhibition (ID1) of p16^INK4a, a tumor suppressor gene operating in the same pathway as Rb

ID2 promoter

E E

ID2

Rb-dependent Rb-dependent cell cell cyclecycle arrestarrest

bHLH Ets Pax

??

antimitogenic

antimitogenic signalssignals

Normal developmentNormal development

T

Myc

T

ID2

Rb

X T

^ID2 ^bHLH^Ets

Pax

??

Cell Cell proliferationproliferation mitogenic

mitogenic signalssignals ( (oncogenesisoncogenesis))

Cancer Cancer

ID2 ID2 ID2

ID2

T

T X

^ID2 ^ID2

ID2 ID2

Myc Myc

Myc Myc

(40)

Other inhibitors of Rb are:

• SV40 large T SV40 large T antigenantigen

• Ad E1A

• human papillomavirus E7

Rb

ID2 Protein-protein recognition: ID2-HLH motif Rb-pocket domain ID2/Rb > 6.5 => Rb inhibition

E2F,

E2F, D-typeD-type cyclinscyclins, , MyoD and MyoD and myogeninmyogenin are are protein-protein protein-protein interaction interaction partners ofpartners of Rb in

Rb in thethe cellcell cyclecycle

A box B box SV40

(41)

Rb binding site

A-B interface (suggested) B box cyclin fold (suggested) B box cyclin fold (suggested) B box cyclin fold (X-ray)

B box cyclin fold (X-ray)

Protein

E2F ID2 E1A

E7 peptide SV40

Binding motif

18-residues HLH

LxCxE LxCxE

LxCxE, helices α2 and α4

A box B box

T antigen

α2

α4 LxCxE

(42)

(43)

A. by expression in a host cell system

B. by chemical ligation of previously synthesized short peptides

E. Fischer (1905): „My entire yearning is directed toward the first synthetic enzyme. If its preparation falls into my lap with the synthesis of a natural protein material, I will consider my mission fulfilled“.

APPLICATION OF THE SCIENCE OF CHEMISTRY TO THE STUDY OF PROTEINS

NOVEL PROTECTING GROUPS NOVEL ACTIVATION METHODS POLYMER-SUPPORTED SYNTHESIS

(44)

Year

Size of peptide chain

200 AA

100 AA

Protein functional domains

smallest proteins

1906 1932 1962 1992

Cbz

SPPS Chemical ligation

Cbz or Z: Carbobenzoxy as protecting group of the α-amino group of the amino acid. Cleavage by catalytic hydrogenolysis

SPPS: Solid Phase Peptide Synthesis

(45)

Bruce Merrifield

Nobel Price in Chemistry in 1984

„One day I had an idea about how the goal of a more efficient synthesis might be achieved. ...“

ClCH₂ Resin

COO^- Cs⁺ CH

BocHN

R(PG)

+

1. Boc deprotection

2. AA coupling n cycles

CH₂ Resin

COO CH

HN

R(PG) Boc: (CH₃)C-O-CO-

Cleavage: diluted TFA

CH₂ Resin

COO CH

BocHN

R(PG)

(46)

Louis Carpino

Y Resin +

n cycles

CO CH HN

R(PG) O

O

Fmoc: 9-fluorenylmethyloxycarbonyl Cleavage: diluted base (piperidine)

Y: NH₂, OH PG: t-Bu, Boc

1. Preactivation or in situ coupling 2. Fmoc deprotection

Resin

TFA

COY CH

HN R COOH

CH FmocHN

R(PG)

(47)

(48)

(49)

Principle of chemoselective reaction applied to unprotected peptide segments

+

Peptide 1

Y

COO^- X

+H₃N

Peptide 2

X

+H₃N

Y

COO^-

Thioester oxime thioether

disulfide thiazolidine peptide bond

(50)

+

Peptide 1:

C-terminal thioester

COO^- C-SR

+H₃N

Peptide 2:

N-terminal cysteine

+H₃NCHCO

Science (1994) 266: 776-79

=^O ^HS

NH

O S

H₂N

O R

+H₃N

COO^-

water pH 7

Thioester-linked intermediate

+H₃N

COO^- NH

O HN

R O

SH

(51)

Microbial ribonuclease (110 AA)

JACS (1997) 119: 4325-29

Zn²⁺

(1-48)-ααCOS-benzyl +

[C⁴⁹]-(49-110)

[C⁴⁹, H⁸⁰, A¹⁰²]-(49-110)

(52)

1. Pseudoproline ligation via imine capture for NT- Cys, NT-Ser, NT-Thr

O O

H O

HX R H₂N

O

O N

O

HX R

O

HN O X

O

R

N X

O

HOH₂C R

O

N + C ^N ^C

N C N C

X = S, O

SR O

HS H₂N

O

S O H₂N

O

O NH

SH

O

O NH

SCH₃

O

N + C

N

C

N C N C

pH > 7

methylation

2. Methionine Ligation

(53)

O

S X HN

R´´

HS X NH

R´´

SR´

O

3. Cysteine mimetic ligation (use of a removable auxiliary)

N + C ^N

C

N C N C

X-SH = HSCH₂CH₂O-

+ ^R´SH

pH > 7

N O

X SH

R´´ - X-SH ^O

NH R´´

SH

CH₃O

SH

CH₃O

SH CH₃O

1-phenyl-2-mercaptoethyl auxiliaries benzyl thiol

auxiliary mercaptoethoxy

auxiliary

(54)

Method 3:

Chemical ligation using 1-(4-methoxyphenyl)-2- mercaptoethyl removable auxiliary

PNAS (2001) 98, 6554-59

(55)

NH

O O

HN N

H₂N N

N

O O

H₂N N

NH

O SSR

O

4. Histidine ligation

N + C ^N _C

N C

NH R´´

O O

O SR´

5. Direct amide bond formation

H₂N R´´

O

N + C ^N ^C

Boc-HN NH-Boc

slow fast thiophenol

Thiophenol/Cu⁺/Cu⁺⁺

R. Ingenito, unpublished results

(56)

A. Noncoded Amino Acids: Chemically or post-translationally modified.

B. Precise Covalent Modification: Substitution of a natural structural motif with a building block (i. e., substitution of the ß-turn Gly-Gly with a sterically constrained bicyclic compound, a rigid mimetic of type II´ ß- turn).

C. Site-Specific Tagged Proteins: Introduction of fluorescent or spin labels, and of affinity tags (e.g., biotin).

D. Backbone Engineering: Replacement of the amide bond (e.g., ester bonds).

E. Structural Biology: „Facile“ access to „large“ amounts of high-purity products can be of great utility for studies of protein structure by NMR and X-ray crystallography.

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• Peptide synthesis:

• Jakubke, H.-D.: Peptide. Spektrum Akademischer Verlag. 1996.

• Greene, T.W, Wuts, P.G.M..Protective groups in organic synthesis. John Wiley and Sons. 1999.

3) Fields, G.B., Noble, R.L.. Solid phase peptide synthesis utilizing 9-

fluorenylmethoxycarbonyl amino acids. Int. J. Pept. Prot. Res. 35, 1990, 161.

4) Tam, J.P., Xu, J., Eom, K.D.. Methods and strategies of peptide ligation. Biopolymers (Pept. Sci.) 60, 2001, 194.

• Dawson, P.E., Kent, S.B.H.. Synthesis of native proteins chemical ligation. Annu. Rev.

Biochem. 69, 2000, 923.

• ID proteins and protein-domain dimerizazion

• Benezra, R., Davis, R.L., Lockshon, D., Turner, D.L., Weintraub H.. The protein Id: a negative regulator of helix-loop-helix DNA binding proteins. Cell 61, 1990, 49.

• Yokota, Y., Mori, S.. Role of Id family proteins in growth control. J. Cell. Physiol. 190, 2002, 21.

3) Klemm, J.D., Schreiber, S.L., Crabtree, G.R.. Dimerization as a regulatory mechanism in signal transduction. Annu. Rev. Immunol. 16, 1998, 569.