the incorporation of unnatural amino acids into proteins by nonsense suppression jason k. pontrello...
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The Incorporation of Unnatural Amino AcidsInto Proteins by Nonsense Suppression
Jason K. PontrelloOctober 25th, 2001
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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
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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
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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
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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
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Translation of Proteins
GTP
elongation factor Tu
ATP
aminoacyl tRNAsynthetase
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Translation Termination
Amber (UAG), Opal(UGA), Ochre(UAA)
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Yeast tRNAPhe and Human eRF1 Release Factor
Bertram, G.; Innes, S.; Minella, O.; Richardson, J. P.; Stansfield, I. Microbiology 2001, 147, 255-269.
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Nonsense Mutation
nonsensemutation
• No corresponding tRNA to continue normal translation of protein
• Causes truncated protein products
• Protein products are usually not functional
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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
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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
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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
+
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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
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Misacylation of Suppressor tRNAs
in vivo
suppressor tRNA
aminoacyl tRNA synthetase
amino acid
chemical
suppressor tRNA(-CA)
pdCpA-amino acid
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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
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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+
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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)
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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
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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
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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
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Selection of Suppressor tRNA for Chemical Misacylation
• Not acylated by endogenous synthetases
• High Suppression Efficiency
UnnaturalAmino acid
IncorporatedInto protein
reacylation
none
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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
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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
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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
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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
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Misacylation of Suppressor tRNAs
chemical
suppressor tRNA(-CA)
pdCpA-amino acid
in vivo
suppressor tRNA
aminoacyl tRNA synthetase
amino acid
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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.
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Mutation Sites to Generate Suppressor tRNATyr Library
Wang, L.; Schultz, P. G. Chem. & Biol. 2001, 8, 883-890.
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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
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Applications of Nonsense Suppression
1. Receptor/Ligand Interactions
2. Biophysical Probes
3. Caged Amino Acids
4. Protein Structure/Function Relationships
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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.
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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
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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.
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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.
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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
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Fluorescence Resonance Energy Transfer (FRET)for Investigating Receptor/Ligand Interactions
sensitizedemission
FRET
donoremission
GFP(donor)
absorbanceemission
emission
tag(acceptor)
absorbance
+
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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)
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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.
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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)
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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
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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
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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
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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 +
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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
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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
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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
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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
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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
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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)
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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.
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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.
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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
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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
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Thanks
• Kiessling Group
• 3rd years Jen , Val, Whitney, Margaret, Chris
• Periodic Table tie for holding up my pants