The Career of Tom W. Muir Scott Simonovich MacMillan Group Meeting February 24 th 2010
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
Cotton, G. J.; Ayers, B.; Xu, R.; Muir, T. W. J. Am. Chem. Soc. 1999, 121, 1100.
NH
HN
ONH
OSR'
O
RGEIKGR
HS
HN
SO O
Me2N
oligopeptide PG
fluorophore
ligation site
Chemical Ligation
Cotton, G. J.; Ayers, B.; Xu, R.; Muir, T. W. J. Am. Chem. Soc. 1999, 121, 1100.
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
Cotton, G. J.; Ayers, B.; Xu, R.; Muir, T. W. J. Am. Chem. Soc. 1999, 121, 1100.
■ 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
Expressed Protein Ligation (EPL)
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
Expressed Protein Ligation (EPL)
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)
Expressed Protein Ligation (EPL)
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
Expressed Protein Ligation (EPL)
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
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
peptidepeptidepeptide
peptidepeptide
peptide
■ Dramatic changes in primary structure lead to changes in function
Conditional Protein Splicing
Small Molecule 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)
Small Molecule Protein Splicing
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
Small Molecule Protein Splicing
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?
Small Molecule Protein Splicing
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
Small Molecule Protein Splicing
peptide peptide peptidepeptide
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
Small Molecule Protein Splicing
peptide peptide peptidepeptide
peptide
peptide
FKBP12 FRB
VMAN VMAC
DnaEN
DnaEC
c-Crk-II protein
peptide peptide
peptide
TCEP (rapamycin)
(rapamycin)
(TCEP)P(CH2CH2CO2H)3
Small Molecule Protein Splicing
peptide peptide peptidepeptide
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