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The Incorporation of Unnatural Amino AcidsInto Proteins by Nonsense Suppression
Jason K. PontrelloOctober 25th, 2001
Outline
1. Receptor/Ligand Interactions
2. Biophysical Probes
3. Caged Amino Acids
4. Protein Structure/Function Relationships
1. Chemically Misacylated Suppressor tRNAs
2. In Vivo Misacylated Suppressor tRNAs
Nonsense Suppression Methodology
Applications
Methods for Unnatural Amino Acid Incorporation into Proteins
• synthesis
• tRNA selection
• tRNA modification
• selection process
Methods for Incorporation of Unnatural Amino Acids into Proteins
• Total chemical synthesis (greatest freedom in residues)
• Post-translational modification by chemical and enzymatic means
Largely limited to 30-50 residues
• Native chemical ligation of fragments
Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. H. Science 1997, 266, 776-779.
HN
R1
SR'
OH2N
HS
NH
O
HN
R1
S
OH2N
NH
O
HN
R1
NH
OHS
HN
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
• Unnatural nucleotides (codon/anticodon pair)
Ma, C.; Kudlicki, W.; Odom, O. W.; Kramer, G.; Hardesty, B. Biochemistry 1993, 32, 7939-7945.
Bain, J. D.; Switzer, C.; Chamberlin, A. R.; Benner, S. A. Nature 1992, 356, 537-539.
ON
N
NH H
O
H
H
O
N
NN
H
NN
OHO
PO O
O
O
P
O
OO
O
OHO
P
O
O
O
OP
OO
O
ON
N
OH
N
H
H
N
O
NN
NN
OHO
PO O
O
O
P
O
OO
O
OHO
P
O
O
O
OP
OO
O
H
H
C-G pair isoC-isoG pair
Nonsense Suppression
Advantages
Limitations
• Ability to selectively incorporate a single unnatural amino acid at a specific site in a protein
• Can be used in vitro or in vivo
• Only works for -amino acids
• Cannot be used to incorporate D-amino acids
• Efficiency of incorporation is variable and not well understood
Translation of Proteins
GTP
elongation factor Tu
ATP
aminoacyl tRNAsynthetase
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
nonsensemutation
• 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
Shih, L. B.; Bayley, H. Anal. Biochem. 1985, 144, 132-141.
“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.”
CO2H
NH2
F3C NN
6
Nonsense Suppression Methodology
Noren, C. J.; Anthony-Cahill, S. J.; Griffith, M. C.; Schultz, P. G. Science 1989, 244, 182-188.
Site-directedmutagenesis
transcription
translation
In vitro and in vivo Systems to Produce Protein
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.
translationalmixture
Xenopusoocyte
in vitro in vivo
+
Misacylation of tRNAs
• The first report
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.
cysteine-tRNACys alanine-tRNACys
chemicaldesulfurization
Misacylation of Suppressor tRNAs
in vivo
suppressor tRNA
aminoacyl tRNA synthetase
amino acid
chemical
suppressor tRNA(-CA)
pdCpA-amino acid
Suppressor tRNA(-CA) Synthesis 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.
• Termination by mRNA hairpin loop formation
• Termination by runoff transcription
Overview Chemical Misacylation of Suppressor tRNAs
Gilmore, M. A.; Steward, L. E.; Chamberlin, A. R. Topics Curr. Chem. 1999, 202, 77-99.
suppressor tRNA(-CA)
ONO
O
P OO
O
O
O
P
O
O
O
N
O
NH2
N
NN
N
NH2
OHO
NH2
R
pdCpA-amino acid
T4 ligase+
Synthesis of pdCpA
Robertson, S. A.; Noren, C. J.; Anthony-Cahill, S. J.; Griffith, M. C.; Schultz, P. G. Nuc. Acid Res. 1989, 17(23), 9649-9660.
ONdMTrO
O
P
N
O
NHBz
NCCH2CH2O N
ONdMTrO
O
P ONCCH2CH2O
O
O
BzO
N
O
NHBz
N
NN
N
NBz2
OBz
ONO
O
P ONCCH2CH2O
O
O
BzO
N
O
NHBz
N
NN
N
NBz2
OBz
P
O
NCCH2CH2O
NCCH2CH2OONO
O
P OO
O
O
HO
P
O
O
O
N
O
NH2
N
NN
N
NH2
OH
1. A-Bz4, tetrazole
2. I2, THF/H2O
(76% yield)
conc. NH4OH
(used crude)
1. TsOH2. (iPr)2NP(OCH2CH2CN)2, tetrazole3. I2, THF/H2O
(80% yield)
Acylation of pdCpA with Amino Acid
Robertson, S. A.; Ellman, J. A.; Schultz, P. G. J. Am. Chem. Soc. 1991, 113, 2722-2729.
ONO
O
P OO
O
O
HO
P
O
O
O
N
O
NH2
N
NN
N
NH2
OH
NH
PGR
O
O
CN
DMF, nBu4N+OAc-
(76-87% yield)
ONO
O
P OO
O
O
O
P
O
O
O
N
O
NH2
N
NN
N
NH2
O
O HN
R
PGH
Ligation of pdCpA-aa to tRNA(-CA)
Heckler, T. G.; Chang, L.-H.; Zama, Y.; Naka, T.; Chorghade, M. S.; Hecht, S. M. Biochemistry 1984, 23, 1468-1473.
ONO
O
P OO
O
O
O
P
O
O
O
N
O
NH2
N
NN
N
NH2
O
O HN
R
PGH
ONO
O
P OO
O
O
O
P
O
O
O
N
O
NH2
N
NN
N
NH2
O
O HN
R
PGH
tRNA
tRNA-C OH
T4 RNA ligase
deprotect
ONO
O
P OO
O
O
O
P
O
O
O
N
O
NH2
N
NN
N
NH2
O
O NH2
RH
tRNA
Misacylation of tRNAs: Protecting Groups for Amino Acids
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.
6-Nitroveratryl oxycarbonyl (NVOC)
4-Pentenoyl
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.
H3CO
H3CO
N
OHN
O
O
O
R1
O H
N
O
OCH3
H3CO
hv = 350nm
1mM KOAc, pH 4.5+
O
O
tRNAH2N
O
O
R1
tRNA
OHN
O
O
O
R1
I2, H2O
( 92% yield)+tRNA H2N
O
O
R1
tRNAO
O
I
Selection of Suppressor tRNA for Chemical Misacylation
• Not acylated by endogenous synthetases
• High Suppression Efficiency
UnnaturalAmino acid
IncorporatedInto protein
reacylation
none
Selection of Suppressor tRNA
Fersht, A. R.; Dingwall, C. Biochemistry 1979, 18, 2627-2631.
• No “double-sieve” editing for glycyl-tRNA synthetases
• Two base pair changes in acceptor stem:
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
Optimal T7 RNA polymerase promoter into the DNA template
Eliminated recognition for E. coli Gly synthetase
Selection of Suppressor tRNA
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.
• 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
Selection of Suppressor tRNA
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.
• Naturally introduces Glutamine at UAG codon
• Modified acceptor stem mutants (THG73 and THA73)
• Good suppression in vivo and in vitro
Selection of Suppressor tRNA
Cload, S. T.; Liu, D. R.; Froland, W. A.; Shultz, P. G. Chem. & Biol. 1996, 3, 1033-1038.
• E. coli tRNAAsnCUA and T. thermophila tRNAGln
CUA best overall
• Suppression Efficiency subject to variables not understood:Different proteins and different sites can give varied results
T4 lysozyme at site 82 Chorismate mutase at site 88
Misacylation of Suppressor tRNAs
chemical
suppressor tRNA(-CA)
pdCpA-amino acid
in vivo
suppressor tRNA
aminoacyl tRNA synthetase
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
Wang, L.; Schultz, P. G. Chem. & Biol. 2001, 8, 883-890.
• orthogonal tRNAs• non-functional tRNAs
Toxic barnase tRNA library endogenous
negativeselection
-lactamase endogenoustRNA library
synthetasepositiveselection
ampicillin• orthogonal tRNA/ synthetase pair
M. jannaschii E. coli
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
Li, L.; Zhong, W.; Zacharias, N.; Gibbs, C.; Lester, H. A. Dougherty, D. A. Chem & Biol 2001, 8, 47-58.
Acetylcholine (ACh)O
O
NMe3
NH
O
OCH2CH2CN
O
NMe3
NVOCNH
O
OCH2CH2CN
O
CMe3
NVOC
n 3
n = 2,3,4,5
Probing Activity of nAChR with Tethered Agonists
NH
O
O
NMe3n
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
sensitizedemission
FRET
donoremission
GFP(donor)
absorbanceemission
emission
tag(acceptor)
absorbance
+
Receptor/Ligand Interactions:Neurokinin (Tachykinin)-2 Receptor (NK2)
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.
Probe
548nm572nm
Emission550nm
Abs476nm
Antagonist ligand
NH
CO2H
NH2
NON
O2N
3-N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)-2,3- diamino-propionic acid (NBD-Dap)
PhCO-K(TMR)-A-DW-F-DP-P-Nle-NH2
(TMR = tetramethylrhodamine)
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
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.
-galactosidase at 340nm excitation
dnsLys (13.6 nM, —)Phe (21.4 nM, ----)wild-type (10.5 nM, . . . . )
NH
CO2H
NH2
HO
NH
N
CO2H
NH2
S
HN CO2H
NH2OO
N
5-Hydroxytryptophan(5-OHTrp)
7-Azatryptophan(7-azaTrp)
-Dnsyllysin(dnsLys)
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.
Biophysical Spin Labeled Probes
CO2H
NH2
S
N
O
CO2H
N
O
NH2
CO2H
NH2
NO
TOAC
T4 Lysozyme (pmole)Ser44 to spin label
X-band EPR spectrum
Caged Amino Acids:Caged Tyrosine to Investigate Membrane Trafficking
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.
O(H)
PO3-2
Mus musculus Kir 2.1 inwardly rectifying K+ channel
kinaseATP ADP
or O(H)
O2N
PO3-2
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
kinaseATP ADP
NH
O
NO2
O
NH
OH
NOO
H
O
+
hv300-350 nm
Protein Structure/Function Relationship:Photochemical Protein Backbone Cleavage
England, P. M.; Lester, H. A.; Davidson, N.; Dougherty, D. A. Proc. Natl. Assoc. Sci. 1997, 94, 11025-11030.
o-Nitrophenyl Glycine (Npg)
NH
R1HN
O
NH
O R2
ONO2
NH2
O
NH
R1
NO
NHO
O R2
O
hv +
England, P. M.; Lester, H. A.; Davidson, N.; Dougherty, D. A. Proc. Natl. Assoc. Sci. 1997, 94, 11025-11030.
Drosophila Shaker B K+ ion channel
Protein Structure/Function Relationship:Photochemical Protein Backbone Cleavage
England, P. M.; Lester, H. A.; Davidson, N.; Dougherty, D. A. Proc. Natl. Assoc. Sci. 1997, 94, 11025-11030.
Nicotinic Acetylcholine Receptor (nAChR)
Protein Structure/Function Relationship:Photochemical Protein Backbone Cleavage
Protein Structure/Function Relationship:Firefly Luciferase
Mamaev, S. V.; Laikhter, A. L.; Arslan, T.; Hecht, S. M. J. Am. Chem. Soc. 1996, 118, 7243-7244.
HO S
N N
S
CO2H
O S
N N
S
O
luciferin Oxyluciferine dianion
Protein Structure/Function Relationship:Firefly Luciferase
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.
NH
O
HO
Wild-type Serine (584nm) Serine Phosphonate (584nm)
NH
O
PO O
O
Glycosyl Serine (584nm)
NH
O
OO
OH
OH
HOHO
NH
O
OP
O
OO
Tyrosine Phosphate (593nm)
NH
O
PO
OO
Tyrosine Phosphonate (603nm)Tyrosine (613nm)
NH
O
HO
Protein Structure/Function Relationship:Tyrosine and Proline Analogs in Adenylate Kinase
Zhao, Z.; Liu, X.; Shi, Z.; Danley, L.; Huang, B.; Jiang, R.-T.; Tsai, M.-D. J. Am. Chem. Soc. 1996, 118, 3535-3536.
NH2
CO2H
HONH2
CO2H
Tyrosine(Tyr)
2,5-Dihydrophenylalanine(DiHPhe)
Tyr-95 does not have to be aromatic
Pro-17 can bemore flexible,but not less (Aze)
NH
CO2H
NH
CO2H
NH
CO2H
NH
CO2H
NH
CO2H
Proline(Pro)
3,4-Dehydroproline (DHP)
Pipecolic Acid(Pip)
Homopipecolid Acid(HPip)
Azetidine 2-Carboxylic Acid(Aze)
MgATP + AMP MgADP + ADP
Protein Structure/Function Relationship: -Branched Amino Acids in T4 Lysozyme -Helix
Cornish, V. W.; Kaplan, M. I.; Veenstra, D. L.; Kollman, P. A.; Schultz, P. G. Biochemistry 1994, 33, 12022-12031.
ADBA destabilizes at Ser44 and stabilizes at Asn68
Molecular dynamics: disruption of helix, but stabilizing hydrophobic packing
• Can destabilize by restriction of rotation
• Can stabilize by improved side-chain van der Waals interactions
CO2H
NH2
CO2H
NH2
CO2H
NH2
CO2H
NH2
CO2H
NH2
CO2H
NH2
L-Alanine(Ala)
L-2-Aminobutanoic Acid(ABA)
L-2-Aminopentanoic Acid(APA)
L-2-Aminohexanoic Acid(AHA)
L-Valine(Val)
L-2-Amino-3,3-Dimethylbutanoic Acid(ADBA)
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.
H2N CO2H
O
OCH2CH=CH2
H2N CO2H
O
OCH2CH=CH2H3C
H3C
H2N CO2H
O
OCH3
H2N CO2H
O
OH
H2N CO2H
O
OHH3C
H2N CO2H
O
OHH3C
H2N CO2H
O
OHH3C
H3C
H2N CO2H
O
OHH3C
H2N CO2H
SO3H
NH
CO2H
O
OH
H3CNH
CO2H
O
OH
H2N CO2H
PO3H2
Aspartic Acid Analogs
Short, G. F. III; Laikhter, A. L.; Lodder, M.; Shayo, Y.; Arslan, T.; Hecht, S. M. Biochemistry 2000, 39, 8768-8781.
Short, G. F. III; Laikhter, A. L.; Lodder, M.; Shayo, Y.; Arslan, T.; Hecht, S. M. Biochemistry 2000, 39, 8768-8781.
4.7 Å
7.7 Å
7.8 Å
7.0 Å
4.1 Å
8.0 Å
Val 82
Ile 84
Asp 25(+deriv.) aspartic acid
H2N CO2H
O
OH
erythro29% increase
H2N CO2H
O
OHH3C
4.2 Å
5.3 Å
7.8 Å
6.2 Å
4.3 Å
7.0 Å
threo87% decrease
H2N CO2H
O
OHH3C
dimethylno activity
H2N CO2H
O
OHH3C
H3C
Binding Pockets of HIV Protease by Molecular Dynamics
Conclusion
Nonsense Suppression Applications:
• Receptor/Ligand Interactions
• Biophysical Probes
• Caged Amino Acids
• Protein Structure/Function Relationships
Unnatural Amino Acids
Phosphorylated/glycosylated Proline derivatives
Fluorescent/spin labels Tethered agonists
Thanks
• Kiessling Group
• 3rd years Jen , Val, Whitney, Margaret, Chris
• Periodic Table tie for holding up my pants
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