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The Incorporation of Unnatural Amino Acids
Into Proteins by Nonsense Suppression
Jason K. Pontrello
October 25th, 2001
Outline
Methods for Unnatural Amino Acid Incorporation into Proteins
Nonsense Suppression Methodology
1. Chemically Misacylated Suppressor tRNAs
• synthesis
• tRNA selection
2. In Vivo Misacylated Suppressor tRNAs
• tRNA modification
• selection process
Applications
1. Receptor/Ligand Interactions
2. Biophysical Probes
3. Caged Amino Acids
4. Protein Structure/Function Relationships
Methods for Incorporation of Unnatural Amino Acids into Proteins
• Total chemical synthesis (greatest freedom in residues)
Largely limited to 30-50 residues
• Native chemical ligation of fragments
O
H
N
H 2N
SR'
R1
O
O
N
H
H 2N
O
H
N
S
HS
R1
O
N
H
HS
H
N
H
N
N
H
R1
Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H. Science 1997, 266, 776-779.
• Post-translational modification by chemical and enzymatic means
O
Methods for Incorporation of Unnatural Amino Acids into Proteins
• In vivo - growth in unnatural amino acid
• In vitro - modification lysine/cysteine already acylated to tRNA
• 4 base codons
Ma, C.; Kudlicki, W.; Odom, O. W.; Kramer, G.; Hardesty, B. Biochemistry 1993, 32, 7939-7945.
• Unnatural nucleotides (codon/anticodon pair)
O
O
OH P O
O
N
H
O
O P O
O
N
H
N
N
N
O
H
O
N
H
N
N
H
H
O
O
O
O
P
O
O
O OH
O P O
O
O
O
O P O
O
N
N
O
C-G pair
H
O OH
O P O
O
O
OH P O
O
N
N
N
H
N
N
H
N
O
O
O
O
O
P
O
H
isoC-isoG pair
O
Bain, J. D.; Switzer, C.; Chamberlin, A. R.; Benner, S. A. Nature 1992, 356, 537-539.
Nonsense Suppression
Advantages
• Ability to selectively incorporate a single unnatural
amino acid at a specific site in a protein
• Can be used in vitro or in vivo
Limitations
• Only works for a-amino acids
• Cannot be used to incorporate D-amino acids
• Efficiency of incorporation is variable and not
well understood
Translation of Proteins
ATP
GTP
elongation
factor Tu
aminoacyl tRNA
synthetase
Translation Termination
Amber (UAG), Opal(UGA), Ochre(UAA)
Yeast tRNAPhe and Human eRF1 Release Factor
Bertram, G.; Innes, S.; Minella, O.; Richardson, J. P.; Stansfield, I. Microbiology 2001, 147, 255-269.
Nonsense Mutation
nonsense
mutation
• No corresponding tRNA to continue normal translation of protein
• Causes truncated protein products
• Protein products are usually not functional
The First Suggestion to Use Nonsense Suppression
“Our long-term goal is to introduce 6 at specific sites in
polypeptides during in vitro protein synthesis. Specifically,
we intend to chemically acylate suppressor tRNAs and
introduce the diazirine at amber mutation sites.”
F 3C
N
N
6
CO2H
NH2
Shih, L. B.; Bayley, H. Anal. Biochem. 1985, 144, 132-141.
Nonsense Suppression Methodology
Site-directed
mutagenesis
transcription
translation
Noren, C. J.; Anthony-Cahill, S. J.; Griffith, M. C.; Schultz, P. G. Science 1989, 244, 182-188.
In vitro and in vivo Systems to Produce Protein
+
in vitro
translational
mixture
in vivo
Xenopus
oocyte
Thorson, J. S.; Cornish, V. W.; Barrett, J. E.; Cload, S. T.; Yano, T.; Schultz, P. G. Methods Mol. Biol. 1998, 77, 43-73.
Dougherty, D. A. Curr. Opin. Chem. Biol. 2000, 4, 645-652.
Misacylation of tRNAs
• The first report
chemical
desulfurization
cysteine-tRNACys
alanine-tRNACys
Chapeville, F.; Lipmann, F.; von Ehrenstein, G.; Weisblum, B.; Ray, W. J.; Benzer, S. Proc. Natl. Acad. Sci. 1962, 48, 1086-1092.
von Ehrenstein, G.; Weisblum, B.; Benzer, S. Proc. Natl. Acad. Sci. 1963, 49, 669-675.
• Significant contributions by Sidney M. Hecht
Hecht, S. M. Acc. Chem. Res. 1992, 25(12), 545-552.
Misacylation of Suppressor tRNAs
chemical
pdCpA-amino acid
suppressor tRNA(-CA)
in vivo
aminoacyl tRNA
synthetase
suppressor tRNA
amino acid
Suppressor tRNA(-CA) Synthesis by Runoff Transcription
• Termination by mRNA hairpin loop formation
• Termination by runoff transcription
Uhlenbeck, O. C.; Gumport, R. I. The Enzymes, 1982, 15, 31-58.
Silber, R.; Malathi, V. G.; Hurwitz, J. Proc. Natl. Acad. Sci. 1972, 69, 3009-3013.
Overview Chemical Misacylation of Suppressor tRNAs
NH2
N
O
O P O
N
O
NH2
O
+
O
O
N
O P O
O
N
O
N
T4 ligase
N
O
O
OH
R
suppressor tRNA(-CA)
NH2
pdCpA-amino acid
Gilmore, M. A.; Steward, L. E.; Chamberlin, A. R. Topics Curr. Chem. 1999, 202, 77-99.
Synthesis of pdCpA
NHBz
NHBz
N
N
dMTrO
N
dMTrO
O
NCCH2 CH2O
O
P
N
1. A-Bz4, tetrazole
2. I 2, THF/H 2O
N
NBz2
O
NCCH2 CH2O P O
O
N
N
O
NCCH2 CH2O P O
NCCH2 CH2O
O
O
NH2
N
N
O
HO
1. TsOH
2. (iPr)2NP(OCH2CH2CN) 2,
tetrazole
3. I 2, THF/H 2O
OBz
NHBz
NH2
O
O P O
O
N
O
BzO
N
N
N
(76% yield)
O
O P O
O
O
O
O
N
N
conc. NH4OH
N
N
O
NBz2
O
NCCH2 CH2O P O
O
N
N
O
(used crude)
OH
( 80% yield)
O
BzO
N
N
OBz
Robertson, S. A.; Noren, C. J.; Anthony-Cahill, S. J.; Griffith, M. C.; Schultz, P. G. Nuc. Acid Res. 1989, 17(23), 9649-9660.
Acylation of pdCpA with Amino Acid
NH2
NH2
N
O
O P O
N
O
R
PG N
O
H
O
NH2
O
O P O
N
O
N
O
HO
OH
N
N
O
O P O
O
CN
O
+
DMF, nBu4N OAc
-
N
O
O
O
NH2
O
O P O
N
O
N
O
N
(76-87% yield)
O
N
N
O
R
H
O
HN PG
Robertson, S. A.; Ellman, J. A.; Schultz, P. G. J. Am. Chem. Soc. 1991, 113, 2722-2729.
Ligation of pdCpA-aa to tRNA(-CA)
NH2
O
O P O
O
tRNA O
N
N
NH2
N
N
O
O
N
tRNA-C
OH
T4 RNA ligase
N
tRNA O
N
O P O
O
O
O
O
O P O
O
NH2
NH2
N
O P O
O
O
O
NH2
O
O P O
O
N
N
O
O
O
N
N
O
NH2
O
O P O
O
N
N
O
O
N
R
H
H
PG
N
O
R
H
HN
deprotect
N
O
O
R
O
N
O
HN
PG
O
NH2
Heckler, T. G.; Chang, L.-H.; Zama, Y.; Naka, T.; Chorghade, M. S.; Hecht, S. M. Biochemistry 1984, 23, 1468-1473.
Misacylation of tRNAs: Protecting Groups for Amino Acids
6-Nitroveratryl oxycarbonyl (NVOC)
O
O
H3CO
N
H
O
O
hv = 350nm
O
H
N
O
H3CO
O
O
tRNA
H 2N
O
1mM KOAc, pH 4.5
N
tRNA
R1
+
H3CO
R1
OCH3
Patchornik, A.; Amit, B.; Woodward, R. B. J. Am. Chem. Soc. 1970, 92, 6333-6335.
Yip, R. W.; Wen, Y. X.; Gravel, D.; Giasson, R.; Sharma, D. K. J. Phys. Chem. 1991, 95, 6078-6081.
4-Pentenoyl
I
O
H
N
O
O
O
O
R1
tRNA
I2, H 2O
H 2N
O
( 92% yield)
tRNA
O
+
O
R1
O
Lodder, M.; Golovine, S.; Laikhter, A. L.; Karginov, V. A.; Hecht, S. M. J. Org. Chem. 1998, 63, 794-803.
Madsen, R.; Roberts, C.; Fraser-Reid, B. J. Org. Chem. 1995, 60, 7920-7926.
Selection of Suppressor tRNA for Chemical Misacylation
• Not acylated by endogenous synthetases
Unnatural
Amino acid
Incorporated
Into protein
reacylation
none
• High Suppression Efficiency
Selection of Suppressor tRNA
• No “double-sieve” editing for glycyl-tRNA synthetases
Fersht, A. R.; Dingwall, C. Biochemistry 1979, 18, 2627-2631.
• Two base pair changes in acceptor stem:
Optimal T7 RNA polymerase promoter into the DNA template
Eliminated recognition for E. coli Gly synthetase
Bain, J. D.; Diala, E. S.; Glabe, C. G.; Wacker, D. A.; Lyttle, M. H.; Dix, T. A.; Chamberlin, A. R. Biochemistry 1991, 30, 5411-5421
Selection of Suppressor tRNA
• Poorly recognized by the E. coli Phe synthetase
• Low suppressor efficiency
• E. coli ribosome affinity reduced for yeast tRNAPhe
• Polar amino acids poorly incorporated
• New suppressor tRNAs
Cload, S. T.; Liu, D. R.; Froland, W. A.; Schultz, P. G. Chem. & Biol. 1996, 3, 1033-1038.
Ellman, J.; Mendel, D.; Anthony-Cahill, S.; Noren, C. J.; Schultz, P. G. Methods in Enzymology 1991, 202, 301-336.
Selection of Suppressor tRNA
• Naturally introduces Glutamine at UAG codon
• Modified acceptor stem mutants (THG73 and THA73)
• Good suppression in vivo and in vitro
Saks, M.; Sampson, J. R.; Nowak, M. W.; Kearney, P. C.; Du, F.; Abelson, J. N.; Lester, H. A.;
Dougherty, D. A. J. Biol. Chem. 1996, 271, 23169-23175.
Cload, S. T.; Liu, D. R.; Froland, W. A.; Shultz, P. G. Chem. & Biol. 1996, 3, 1033-1038.
Selection of Suppressor tRNA
T4 lysozyme at site 82
Chorismate mutase at site 88
• E. coli tRNAAsnCUA and T. thermophila tRNAGlnCUA best overall
• Suppression Efficiency subject to variables not understood:
Different proteins and different sites can give varied results
Cload, S. T.; Liu, D. R.; Froland, W. A.; Shultz, P. G. Chem. & Biol. 1996, 3, 1033-1038.
Misacylation of Suppressor tRNAs
chemical
pdCpA-amino acid
suppressor tRNA(-CA)
in vivo
aminoacyl tRNA
synthetase
suppressor tRNA
amino acid
Requirements for In Vivo Misacylation
• Uptake of Unnatural Amino Acid not toxic to cell
• Suppressor tRNA only acylated by correct synthetase
(orthogonal tRNA/synthetase pair)
Saks, M. E. Proc. Natl. Acad. Sci. 2001, 98, 2125-2127.
Mutation Sites to Generate Suppressor tRNATyr Library
Wang, L.; Schultz, P. G. Chem. & Biol. 2001, 8, 883-890.
Double Selection Screen for Orthogonal tRNA/Synthtase Pair
M. jannaschii
E. coli
negative
selection
Toxic barnase
tRNA library
endogenous
positive
selection
• orthogonal tRNA/
synthetase pair
• orthogonal tRNAs
• non-functional tRNAs
ampicillin
synthetase
b-lactamase
tRNA library
Wang, L.; Schultz, P. G. Chem. & Biol. 2001, 8, 883-890.
endogenous
Applications of Nonsense Suppression
1. Receptor/Ligand Interactions
2. Biophysical Probes
3. Caged Amino Acids
4. Protein Structure/Function Relationships
Receptor/Ligand Interactions:
Nicotinic Acetylcholine Receptor (nAChR)
Li, L.; Zhong, W.; Zacharias, N.; Gibbs, C.; Lester, H. A. Dougherty, D. A. Chem & Biol 2001, 8, 47-58.
Synthesis of Tethered Agonists for nAChR
O
NMe3
O
O
NVOC
OCH2CH2CN
N
H
O
Acetylcholine (ACh)
NMe3
n
O
NVOC
CMe3
3
OCH2CH2CN
N
H
O
n = 2,3,4,5
Li, L.; Zhong, W.; Zacharias, N.; Gibbs, C.; Lester, H. A. Dougherty, D. A. Chem & Biol 2001, 8, 47-58.
Probing Activity of nAChR with Tethered Agonists
O
NMe3
n
N
H
O
n = 2,3,4,5
Li, L.; Zhong, W.; Zacharias, N.; Gibbs, C.; Lester, H. A. Dougherty, D. A. Chem & Biol 2001, 8, 47-58.
Proposed View of nAChR Agonist Binding Site
Li, L.; Zhong, W.; Zacharias, N.; Gibbs, C.; Lester, H. A. Dougherty, D. A. Chem & Biol 2001, 8, 47-58.
Biophysical Probes
• Unnatural amino acids as fluorescence or spin labels
• Uses in:
Protein-protein interactions
Protein-ligand interactions
Sensitive detection
Protein structure determination and conformational changes
Fluorescence Resonance Energy Transfer (FRET)
for Investigating Receptor/Ligand Interactions
GFP
(donor)
emission
sensitized
emission
absorbance
FRET
+
donor
emission
tag
(acceptor)
absorbance
emission
Receptor/Ligand Interactions:
Neurokinin (Tachykinin)-2 Receptor (NK2)
Probe
3-N-(7-Nitrobenz2-oxa-1,3-diazol- O2N
4-yl)-2,3- diaminopropionic acid
(NBD-Dap)
Abs
476nm
Antagonist ligand
N O
N
N
H
Emission
550nm
CO2H
PhCO-K(eTMR)-A-DW-F-DP-P-Nle-NH2
(TMR = tetramethylrhodamine)
NH2
548nm
572nm
Turcatti, G.; Nemeth, K.; Edgerton, M. D.; Meseth, U.; Talabot, F.; Peitsch, M.; Knowles, J.; Vogel, H.;
Chollet, A. J. Biol. Chem. 1996, 271, 19991-19998.
Receptor/Ligand Interactions: NK2
G-Protein Coupled Receptor (7-Transmembrane Receptor)
Turcatti, G.; Nemeth, K.; Edgerton, M. D.; Meseth, U.; Talabot, F.; Peitsch, M.; Knowles, J.; Vogel, H.;
Chollet, A. J. Biol. Chem. 1996, 271, 19991-19998.
Biophysical Probes: Fluorescence
b-galactosidase at 340nm excitation
HO
CO2H
N
H
CO2H
N
NH2
5-Hydroxytryptophan
(5-OHTrp)
N
7-Azatryptophan
(7-azaTrp)
S
O
NH2
N
H
H
N
O
CO2H
NH2
e-Dansyllysine
(dnsLys)
dnsLys (13.6 nM, —)
Phe (21.4 nM, ----)
wild-type (10.5 nM, . . . . )
Steward, L. E.; Collins, C. S.; Gilmore, M. A.; Carlson, J. E.; Ross, J. B. A.; Chamberlin, A. R. J. Am. Chem. Soc. 1997, 119, 6-11.
Biophysical Spin Labeled Probes
CO2H
S
X-band EPR spectrum
NH2
N
O
CO2H
NH2
N
O
O
N
CO2H
NH2
TOAC
T4 Lysozyme (pmole)
Ser44 to spin label
Cornish, V. W.; Benson, D. R.; Altenbach, C. A.; Hideg, K.; Hubbell, W. L.; Schultz, P. G. Proc. Natl. Acad. Sci. 1994, 91, 2910-2914.
Caged Amino Acids:
Caged Tyrosine to Investigate Membrane Trafficking
ATP
ADP
kinase
O(H)
PO3 -2
Mus musculus Kir 2.1 inwardly rectifying K+ channel
Tong, Y.; Brandt, G. S.; Li, M.; Shapovalov, G.; Slimko, E.; Karschin, A.; Dougherty, D. A.; Lester, H. A.
J. Gen. Physiol. 2001, 117, 103-118.
Caged Tyrosine to Investigate Membrane Trafficking
ATP
ADP
kinase
O
NO2
N
H
O
or
hv
300-350 nm
O(H)
PO3 -2
OH
+
N
H
O2N
H
O
NO
O
Tong, Y.; Brandt, G. S.; Li, M.; Shapovalov, G.; Slimko, E.; Karschin, A.; Dougherty, D. A.; Lester, H. A.
J. Gen. Physiol. 2001, 117, 103-118.
Protein Structure/Function Relationship:
Photochemical Protein Backbone Cleavage
R1
O
R2
R1
H
N
N
H
hv
NO2
NH2
N
H
NH
O
O
O
O
+
O
R2
NH
NO
O
o-Nitrophenyl Glycine (Npg)
England, P. M.; Lester, H. A.; Davidson, N.; Dougherty, D. A. Proc. Natl. Assoc. Sci. 1997, 94, 11025-11030.
Protein Structure/Function Relationship:
Photochemical Protein Backbone Cleavage
Drosophila Shaker B K+ ion channel
England, P. M.; Lester, H. A.; Davidson, N.; Dougherty, D. A. Proc. Natl. Assoc. Sci. 1997, 94, 11025-11030.
Protein Structure/Function Relationship:
Photochemical Protein Backbone Cleavage
Nicotinic Acetylcholine Receptor (nAChR)
England, P. M.; Lester, H. A.; Davidson, N.; Dougherty, D. A. Proc. Natl. Assoc. Sci. 1997, 94, 11025-11030.
Protein Structure/Function Relationship:
Firefly Luciferase
HO
N
N
S
S
luciferin
CO2H
O
N
N
S
S
O
Oxyluciferine dianion
Mamaev, S. V.; Laikhter, A. L.; Arslan, T.; Hecht, S. M. J. Am. Chem. Soc. 1996, 118, 7243-7244.
Protein Structure/Function Relationship:
Firefly Luciferase
O
O
O
OH
P
HO
HO
HO
O
O
OH
N
H
N
H
N
H
O
O
O
Wild-type Serine (584nm)
Serine Phosphonate (584nm)
HO
O
O
N
H
P
O
O
O
O
N
H
O
Tyrosine (613nm)
Glycosyl Serine (584nm)
P
O
N
H
O
Tyrosine Phosphate (593nm)
O
Tyrosine Phosphonate (603nm)
Mamaev, S. V.; Laikhter, A. L.; Arslan, T.; Hecht, S. M. J. Am. Chem. Soc. 1996, 118, 7243-7244.
Arslan, T.; Mamaev, S. V.; Mamaeva, N. V.; Hecht, S. M. J. Am. Chem. Soc. 1997, 119, 10877-10887.
Protein Structure/Function Relationship:
Tyrosine and Proline Analogs in Adenylate Kinase
MgATP + AMP
MgADP + ADP
CO2H
CO2H
NH2
HO
Tyrosine
(Tyr)
Tyr-95 does not
have to be aromatic
NH2
2,5-Dihydrophenylalanine
(DiHPhe)
CO2H
Pro-17 can be
more flexible,
but not less (Aze)
CO2H
CO2H
NH
NH
NH
Proline
(Pro)
3,4-Dehydroproline
(DHP)
Pipecolic Acid
(Pip)
CO2H
NH
Homopipecolid Acid
(HPip)
CO2H
NH
Azetidine 2-Carboxylic Acid
(Aze)
Zhao, Z.; Liu, X.; Shi, Z.; Danley, L.; Huang, B.; Jiang, R.-T.; Tsai, M.-D. J. Am. Chem. Soc. 1996, 118, 3535-3536.
Protein Structure/Function Relationship:
b-Branched Amino Acids in T4 Lysozyme a-Helix
• Can destabilize by restriction of rotation
• Can stabilize by improved side-chain van der Waals interactions
CO2H
CO2H
NH2
CO2H
NH2
L-Alanine
(Ala)
NH2
L-2-Aminobutanoic Acid
(ABA)
CO2H
NH2
L-2-Aminohexanoic Acid
(AHA)
L-2-Aminopentanoic Acid
(APA)
CO2H
CO2H
NH2
L-Valine
(Val)
NH2
L-2-Amino-3,3-Dimethylbutanoic Acid
(ADBA)
Molecular dynamics: disruption of helix, but stabilizing hydrophobic packing
ADBA destabilizes at Ser44 and stabilizes at Asn68
Cornish, V. W.; Kaplan, M. I.; Veenstra, D. L.; Kollman, P. A.; Schultz, P. G. Biochemistry 1994, 33, 12022-12031.
Protein Structure/Function Relationship:
HIV Protease and Aspartic Acid Analogs
Short, G. F. III; Laikhter, A. L.; Lodder, M.; Shayo, Y.; Arslan, T.; Hecht, S. M. Biochemistry 2000, 39, 8768-8781.
Aspartic Acid Analogs
O
O
H 3C
OH
H 2N
CO2H
H 2N
O
H 3C
OH
CO2H
CO2H
H 2N
O
OH
OH
H 3C
H 2N
CO2H
CO2H
O
OCH2CH=CH2
CO2H
OCH3
H 2N
CO2H
O
OH
N
H
H 3C
H 3C
H 2N
O
H 3C
CO2H
O
OCH2CH=CH2
H 2N
OH
H 2N
O
O
H 3C
H 3C
CO2H
SO3 H
OH
N
H
CO2H
H 2N
CO2H
PO3 H2
H 2N
CO2H
Short, G. F. III; Laikhter, A. L.; Lodder, M.; Shayo, Y.; Arslan, T.; Hecht, S. M. Biochemistry 2000, 39, 8768-8781.
Binding Pockets of HIV Protease by Molecular Dynamics
4.7 Å
7.0 Å
Val 82
O
Ile 84
O
H 3C
OH
7.8 Å
Asp 25
(+deriv.)
CO2H
H 2N
aspartic acid
7.7 Å
O
H 3C
7.8 Å
H 2N
6.2 Å
H 3C
H 3C
OH
7.0 Å
CO2H
threo
87% decrease
4.3 Å
OH
CO2H
erythro
29% increase
4.1 Å
4.2 Å
5.3 Å
H 2N
8.0 Å
H 2N
O
OH
CO2H
dimethyl
no activity
Short, G. F. III; Laikhter, A. L.; Lodder, M.; Shayo, Y.; Arslan, T.; Hecht, S. M. Biochemistry 2000, 39, 8768-8781.
Conclusion
Nonsense Suppression Applications:
• Receptor/Ligand Interactions
• Biophysical Probes
• Caged Amino Acids
• Protein Structure/Function Relationships
Unnatural Amino Acids
Fluorescent/spin labels
Tethered agonists
Phosphorylated/glycosylated
Proline derivatives
Thanks
• Kiessling Group
• 3rd years Jen , Val, Whitney, Margaret, Chris
• Periodic Table tie for holding up my pants