Download Career of Tom Muir

The Career of Tom W. Muir
Scott Simonovich
MacMillan Group Meeting
February 24th 2010
Previous Methods for Peptide Synthesis
O
support
O
O
NH2
peptide
NHBoc
HO
R
R
R'
R'
H
N
NHBoc
O
Solid phase peptide synthesis (SPPS)
desire polypeptide
cleavage
O
R'
H
N
iterations
R
NH2
O
■ Useful method with significant drawbacks
■ Incorporate unnatural or D-amino acids
■ Each step must be very efficient
35 residue polypeptide:
99% yield per step = 49% IY
95% yield per step = 3% IY
■ Maximum length of ~50 amino acids
■ Can be very time consuming
■ Many peptides not suitable for SPPS
Chemical Ligation
S
O
O
polypeptide 1
H3N
SR
HN
polypeptide 2
RSH
Native Ligation (NL)
■ Coupling of unprotected AA's
O
■ Compatible with side chains
polypeptide 1
S
O
H2N
HN
polypeptide 2
■ Nearly quanitative yield
■ General tool for semi-synthesis
■ Works at physiological pH
O
polypeptide 1
SH
HN
H
N
■ Numerous improvements
polypeptide 2
O
Figure adapted from Muir, T. W. Annu. Rev. Biochem. 2003, 72, 249.
Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H. Science 1994, 266, 776.
Chemical Ligation
■ 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
SO2NH
domain 2
(dansylamide)
fluorescent probe
NMe2
■ Abelson nonreceptor protein tyrosine kinase (Abl)
■ src Homolgy 2 domain (SH3) and src Homolgy domain 2 (SH2)
Cotton, G. J.; Ayers, B.; Xu, R.; Muir, T. W. J. Am. Chem. Soc. 1999, 121, 1100.
Chemical Ligation
oligopeptide PG
HS
RGEIKGR
Me2N
N
H
S
O
H
N
O
O
N
H
SR'
O
H
N
O
ligation site
fluorophore
Cotton, G. J.; Ayers, B.; Xu, R.; Muir, T. W. J. Am. Chem. Soc. 1999, 121, 1100.
Chemical Ligation
HS
RGEIKGR
Me2N
H
N
N
H
S
O
O
O
N
H
HS
SR'
O
Abl-SH2
H2N
H
N
O
O
Dns
Abl-SH3
Abl-SH2
6 M GdmCl, 140 mM NaCl, 200 mM phosphate buffer
1.5 vol% benzylmercaptan, 1.5 vol% thiophenol, 96 hours
Dns
Dns
HS
RGEIKGR
N
H
Factor Xa
Abl-SH2
O
deprotection
HS
O
Abl-SH2
H2N
Abl-SH3
SR'
O
Cotton, G. J.; Ayers, B.; Xu, R.; Muir, T. W. J. Am. Chem. Soc. 1999, 121, 1100.
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?
Cotton, G. J.; Ayers, B.; Xu, R.; Muir, T. W. J. Am. Chem. Soc. 1999, 121, 1100.
Chemical Ligation
■ Chemical modification of proteins + external impulse
■ Semi-synthesis through native ligation with photolabile PG
■ Traditionally difficult to prepare photocaged systems
UV light
polypeptide
protein that controls
cell function
ligation
polypeptide
photorelease
photocaged
protein
cell
■ Control of protein function through subcellular localization with light
Pellois, J-.; Muir, T. W. Angew. Chem. Int. Ed. 2005, 44, 5713.
Chemical Ligation
required for ligation
required for UV
photolytic cleavage
HS
H3N
NO2
H2N
H
N
O
OMe
O
HN
O
NH2
HN
HN
SO2
O
O
O3S
required for fluorescence
spectroscopy
Texas Red
N
O
N
■ Synthesize fluorophore-bearing fragment and ligate to recombinant protein
Pellois, J-.; Muir, T. W. Angew. Chem. Int. Ed. 2005, 44, 5713.
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
O
Abl-SH3
SR
How are protein-bearing thioesters constructed?
Total synthesis
Expression
O
general methods
protein
Expressed Protein Ligation
SR
Expressed Protein Ligation (EPL)
Protein Splicing
SH
O
SH
O
N-Extein
Intein
N
H
C-Extein
N
H
O
■ No sequence requirements
on exteins
NH2
O
NH
O
O
N-Extein
Intein
S
HS
N
H
O
NH2
Intein
SH
C-Extein
O
NH2
NH2
N-Extein
N-Extein
Intein
NH2
N
H
O
NH2
N
H
C-Extein
O
S
O
HS
SH
O
C-Extein
Muir, T. W. Annu. Rev. Biochem. 2003, 72, 249.
Expressed Protein Ligation (EPL)
N-Extein
SH
O
O
Intein
S
N
H
O
NH2
NH2
N-Extein
S
O
Intein
HS
NH2
N
H
O
NH2
O
C-Extein
C-Extein
Expressed Protein Ligation (EPL)
SH
O
Intein
HS
Me
NH2
O
N-Extein
C-Extein
N
H
SPh
HS
peptide
H2N
O
PhSH
N-Extein
SH
O
O
Intein
S
N
H
Me
NH2
SH
O
C-Extein
N-Extein
N
H
peptide
O
■ Access to large proteins
N-Extein
Intein
NH2
Me
■ General method
S
O
HS
O
N
H
C-Extein
■ Post-translational modifications
■ Structure and functions
Expressed Protein Ligation (EPL)
Protein Conformation
■ Trp intrinsic fluorophore - sensitive to local environment
Trp
HN
Trp
R
R
O
HN
Trp
ligand
HN
O
Trp
HN
Trp
Trp
NH
NH
Trp
■ Risks of destabilizing protein or altering function
■ Trp analogs well suited for studying structure (lack of techniques)
Trp
Expressed Protein Ligation (EPL)
Protein Conformation
■ Trp intrinsic fluorophore - sensitive to local environment
Trp
HN
Trp
R
R
O
HN
Trp
O
ligand
HN
Trp
HN
Trp
Trp
NH
NH
Trp
Trp
N
N
■ Risks of destabilizing protein or altering function
■ Trp analogs well suited for studying structure (lack of techniques)
■ Dynamic properties of SH3 domain
SH2
SH3
c-Crk-I
■ Previously studied in isolation
■ Domain-specific biophysical information
Expressed Protein Ligation (EPL)
O
intein
SH2
SH2
RSH
SR
α-thioester coupling partner
recombinant expression
HS
7AW
His-tag
SH3
Xa
7AW
O
HS
SH2
7AW
SH3
SR
H2N
7AW
7AW
H2N
protease
recombinant expression
(E. coli Trp auxotroph)
7AW
SH3
Terminal cys coupling partner
native
Trp
ligation
Trp
7AW
SH2
SH3
7AW
(6 M GdCl)
c-Crk-I
Muir, T. W.; et al. J. Am. Chem. Soc. 2004, 126, 14404.
Small Molecule Protein Splicing
■ Temporal control over protein function with small molecules
■ Turning protein on/off is difficult with standard genetic techniques
Chemical Genetics
ligand
ligand
Mootz, H. D.; Muir, T. W. J. Am. Chem. Soc. 2002, 124, 9044.
Small Molecule Protein Splicing
■ Temporal control over protein function with small molecules
■ Turning protein on/off is difficult with standard genetic techniques
Chemical Genetics
peptide
peptide
peptide
peptide
peptide
peptide
■ Dramatic changes in primary structure lead to changes in function
Conditional Protein Splicing
Mootz, H. D.; Muir, T. W. J. Am. Chem. Soc. 2002, 124, 9044.
Small Molecule Protein Splicing
O
SH
O
N-Extein
SH
O
N
H
Intein
N
H
O
NH
Intein
HS
C-Extein
O
NH2
NH2
SH
O
■ Protein splicing occurs spontaneously
N-Extein
C-Extein
N
H
Conditional Protein Splicing = Protein Splicing + Molecule-Induced Heterodimerization
HO
FKBP12
FRB
MeO
Me
Me
(rapamycin)
N
O
Me
FKBP12
O
HO
O
O
O
O
OH
Me
O
MeO
OMe
Me
FRB
Me
Me
Small Molecule Protein Splicing
Conditional Protein Splicing = Protein Splicing + Molecule-Induced Heterodimerization
FKBP12
FRB
inteinN
inteinC
peptide
peptide
peptide
peptide
protein splicing
■ Low affinity intein fragments
■ Artificially split VMA intein
■ MBP and His peptide fragments
■ Fragemnts expressed in E. coli
■ No reaction without rapamycin
peptide
peptide
Figures adapted from Mootz, H. D.; Muir, T. W. J. Am. Chem. Soc. 2002, 124, 9044.
Small Molecule Protein Splicing
FKBP12
FRB
VMAN
VMAC
MBP
His
MBP
■ 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?
His
Small Molecule Protein Splicing
tandem
splicing
peptide
peptide
peptide
peptide
peptide
Potential difficulties:
■ Intein specificity
■ Timing of splicing events
■ Occurs under physiological conditions
■ Starting materials only required in low concentrations
peptide
Small Molecule Protein Splicing
tandem
splicing
peptide
peptide
peptide
peptide
peptide
peptide
■ Essentially nothing is known about molecular recognition
DnaEN
DnaEC
naturally split intein
■ Why do naturally split intein spontaneously splice?
■ Suggests intrinsic affinity difference between two intein types
Small Molecule Protein Splicing
FKBP12
FRB
VMAN
VMAC
peptide
peptide
(rapamycin)
P(CH2CH2CO2H)3
(TCEP)
DnaEN
DnaEC
peptide
peptide
peptide
c-Crk-II protein
peptide
TCEP
peptide
peptide
peptide
(rapamycin)
Small Molecule Protein Splicing
FKBP12
FRB
VMAN
VMAC
peptide
peptide
DnaEN
DnaEC
(rapamycin)
P(CH2CH2CO2H)3
(TCEP)
peptide
peptide
peptide
c-Crk-II protein
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
Summary
N-Extein
O
O
polypeptide 1
S
O
S
O
Intein
HS
H2N
HN
polypeptide 2
NH2
N
H
O
C-Extein
NH2
Native Chemical Ligation
FKBP12
FRB
VMAN
VMAC
MBP
Expressed Protein Ligation
His
Conditional and Tandem Protein Splicing
MBA
His