the career of tom w. muir - princeton...

The Career of Tom W. Muir

Scott SimonovichMacMillan Group Meeting

February 24th 2010

Previous Methods for Peptide Synthesis

Solid phase peptide synthesis (SPPS)

peptide

O

R

NH2support

O

HONHBoc

R'

O

R

HN

ONHBoc

R'

O

R

HN

ONH2

R'iterationscleavage

desire polypeptide

■ Useful method with significant drawbacks

■ Maximum length of ~50 amino acids■ Incorporate unnatural or D-amino acids

■ Each step must be very efficient

■ Many peptides not suitable for SPPS

35 residue polypeptide:99% yield per step = 49% IY95% yield per step = 3% IY

■ Can be very time consuming

Chemical Ligation

Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H. Science 1994, 266, 776.

Native Ligation (NL)

Figure adapted from Muir, T. W. Annu. Rev. Biochem. 2003, 72, 249.

polypeptide 1polypeptide 2SR

O

HN

O

H3N

S

polypeptide 1

polypeptide 2HN

O

H2N

S

O

polypeptide 1

polypeptide 2

O

HNSH

O

HN

■ Coupling of unprotected AA's

■ General tool for semi-synthesis

■ Works at physiological pH

■ Numerous improvements

■ Compatible with side chains

■ Nearly quanitative yield

RSH

Chemical Ligation

Cotton, G. J.; Ayers, B.; Xu, R.; Muir, T. W. J. Am. Chem. Soc. 1999, 121, 1100.

■ Applications in protein engineering to study biological systems

■ Insertion of synthetic peptide into recombinant protein

■ Biosensor with properties dependent upon state of system

domain 1 domain 2

fluorescent probe

■ Abelson nonreceptor protein tyrosine kinase (Abl)

■ src Homolgy 2 domain (SH3) and src Homolgy domain 2 (SH2)

NMe2

SO2NH

(dansylamide)

Chemical Ligation


NH

HN

ONH

OSR'

O

RGEIKGR

HS

HN

SO O

Me2N

oligopeptide PG

fluorophore

ligation site

Chemical Ligation


NH

HN

ONH

OSR'

O

RGEIKGR

HS

HN

SO O

Me2N

H2N

HS

O

Abl-SH2

6 M GdmCl, 140 mM NaCl, 200 mM phosphate buffer1.5 vol% benzylmercaptan, 1.5 vol% thiophenol, 96 hours

Abl-SH2

Dns

NH

O

RGEIKGR

HS Factor Xa

deprotection Abl-SH2

Dns

H2NO

HS

Abl-SH2

Dns

Abl-SH3 SR'

O

Abl-SH3

Chemical Ligation


■ Kd = 0.123 0.017 µM

■ Monodentate ligands led to insignificant fluorescence increases

■ Useful biosensor for future investigations

■ High affinity, bidentate ligands

■ in vitro screening of combinatorial peptide libraries

■ Characterize protein-protein interactions that regulate Abl function

domain 1 domain 2

fluorescent probe

Can native ligation regulate molecular processes to study protein function?

Chemical Ligation

Pellois, J-.; Muir, T. W. Angew. Chem. Int. Ed. 2005, 44, 5713.

■ Chemical modification of proteins + external impulse

polypeptide

protein that controlscell function

polypeptide

photocagedprotein

■ Semi-synthesis through native ligation with photolabile PG

■ Traditionally difficult to prepare photocaged systems

UV light

cell

■ Control of protein function through subcellular localization with light

ligation photorelease

Chemical Ligation

Pellois, J-.; Muir, T. W. Angew. Chem. Int. Ed. 2005, 44, 5713.

■ Synthesize fluorophore-bearing fragment and ligate to recombinant protein

NO2

H2N

HS

OMeO

HN

O

H3N

OHN

O

NH2HN

OHN

SO2

O3S

ON N

Texas Red

required for ligationrequired for UV

photolytic cleavage

required for fluorescencespectroscopy

Chemical Ligation

■ Photolysis of protein in a cell

■ Dosable manner with low-intensity UV light

■ Valuable if different protein concentrations trigger different responses

■ Photolysis of small molecule can change function of protein

Abl-SH3 SR

O

How are protein-bearing thioesters constructed?

Total synthesis

Expression

general methodsprotein SR

O

Expressed Protein Ligation

Expressed Protein Ligation (EPL)Protein Splicing

NH

OSH

NH

O

O

NH2

SH

N-Extein InteinC-Extein

S

O

NH

O

O

NH2

SH

N-ExteinIntein

C-ExteinNH2

NH

O

O

NH2

S

N-Extein

InteinC-Extein

NH2

HS

O

N-Extein

Intein

C-Extein

NH2

HS

NH

O

O

NH

OSH

Muir, T. W. Annu. Rev. Biochem. 2003, 72, 249.

■ No sequence requirements on exteins

Expressed Protein Ligation (EPL)

S

O

NH

O

O

NH2

SH

N-ExteinIntein

C-ExteinNH2

NH

O

O

NH2

S

N-Extein

InteinC-Extein

NH2

HS

O


S

O

MeNH

OSH

N-ExteinIntein

C-ExteinNH2

MeNH

OS

N-Extein

InteinC-Extein

NH2

HS

O

HS

O

MeNH

OSH

N-ExteinInteinC-Extein

NH2

SPh

H2N

HS

O

peptide

O

N-Extein NH

O

peptide

SH

PhSH

■ Access to large proteins

■ General method

■ Post-translational modifications

■ Structure and functions


Protein Conformation

HN

OHN

NH

R

HN

OHN

NH

R

ligand

Trp

Trp

Trp

Trp

Trp

Trp

Trp

Trp

■ Trp intrinsic fluorophore - sensitive to local environment

■ Risks of destabilizing protein or altering function

■ Trp analogs well suited for studying structure (lack of techniques)


Protein Conformation

HN

OHN

NH

N

R

HN

OHN

NH

N

R

ligand

Trp

Trp

Trp

Trp

Trp

Trp

Trp

Trp

■ Trp intrinsic fluorophore - sensitive to local environment

■ Risks of destabilizing protein or altering function

■ Trp analogs well suited for studying structure (lack of techniques)

SH2 SH3

c-Crk-I

■ Dynamic properties of SH3 domain

■ Previously studied in isolation

■ Domain-specific biophysical information


SH2 SH3

c-Crk-I

Trp

Trp

7AW

7AW

SH2

SH3

intein

recombinant expression

RSH SH2

α-thioester coupling partner

O

SR

His-tag7AW

7AW

recombinant expression(E. coli Trp auxotroph)

Xa

protease

SH37AW

7AW

Terminal cys coupling partner

H2N

HS

SH2O

SRSH3

7AW

7AWH2N

HS nativeligation

(6 M GdCl)

Muir, T. W.; et al. J. Am. Chem. Soc. 2004, 126, 14404.

Small Molecule Protein Splicing

■ Temporal control over protein function with small molecules

Mootz, H. D.; Muir, T. W. J. Am. Chem. Soc. 2002, 124, 9044.

■ Turning protein on/off is difficult with standard genetic techniques

Chemical Genetics

ligand

ligand


■ Temporal control over protein function with small molecules

Mootz, H. D.; Muir, T. W. J. Am. Chem. Soc. 2002, 124, 9044.

■ Turning protein on/off is difficult with standard genetic techniques

Chemical Genetics

peptidepeptidepeptide

peptidepeptide

peptide

■ Dramatic changes in primary structure lead to changes in function

Conditional Protein Splicing


■ Protein splicing occurs spontaneously

NH

OSH

NH

O

O

NH2

SH

N-Extein InteinC-Extein

N-Extein

Intein

C-Extein

NH2

HS

NH

O

O

NH

OSH

Conditional Protein Splicing = Protein Splicing + Molecule-Induced Heterodimerization

FKBP12 FRB

N

OO

OHO

Me

Me

OMe

O

O

Me

HO

MeO

O

Me

MeOH

MeOO

Me

Me

FKBP12 FRB

(rapamycin)


Conditional Protein Splicing = Protein Splicing + Molecule-Induced Heterodimerization

FKBP12 FRB

peptide peptidepeptidepeptide

Figures adapted from Mootz, H. D.; Muir, T. W. J. Am. Chem. Soc. 2002, 124, 9044.

inteinN inteinC

peptidepeptide

protein splicing■ Low affinity intein fragments

■ Artificially split VMA intein

■ MBP and His peptide fragments

■ Fragemnts expressed in E. coli

■ No reaction without rapamycin


FKBP12 FRB

MBP His

VMAN VMAC

HisMBP

■ First example of protein splicing by small molecule

■ MBP and His are model protein

■ No structural or sequence restrictions to exteins

Is this technique limited to the coupling of two peptide fragments?


peptide peptide peptidepeptide

peptide

peptide

tandemsplicing

Potential difficulties:■ Intein specificity

■ Timing of splicing events

■ Occurs under physiological conditions

■ Starting materials only required in low concentrations



peptide

peptide

tandemsplicing

■ Essentially nothing is known about molecular recognition

naturally split intein

DnaEN DnaEC■ Why do naturally split intein spontaneously splice?

■ Suggests intrinsic affinity difference between two intein types

(rapamycin)

(TCEP)P(CH2CH2CO2H)3



peptide

peptide

FKBP12 FRB

VMAN VMAC

DnaEN

DnaEC

c-Crk-II protein

peptide peptide

peptide

TCEP (rapamycin)

(rapamycin)

(TCEP)P(CH2CH2CO2H)3



peptide

peptide

■ Splicing can occur simultaneously or in a stepwise fashion

■ Additional tags and protecting groups for easy isolation purification

■ Proficient with purified fragments or crude cell lysates

■ Streamlined process this great generality

FKBP12 FRB

VMAN VMAC

DnaEN

DnaEC

c-Crk-II protein

Summary

polypeptide 1

polypeptide 2HN

O

H2N

S

O

NH

O

O

NH2

S

N-Extein

InteinC-Extein

NH2

HS

O

FKBP12 FRB

MBP His

VMAN VMAC

HisMBA

Native Chemical Ligation Expressed Protein Ligation

Conditional and Tandem Protein Splicing

the career of tom w. muir - princeton...

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