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Synthesis Of Proteins Containing
Unnatural Amino Acids
Michigan State University
Department of Chemistry
Justas Jancauskas
Why Introduce Unnatural Amino Acids?
• Drug industry: N-methylated proteins are stable to
proteolysis
• Selective protein labeling with antigenic or
fluorophoric tags
• Selective protein posttranslational modification
• Probing protein structure and function relationship
van Maarseveen, J. H.; Back, J. W. Angew. Chem. Int. Ed. 2003, 42, 5926–5928.
Mehl, R. A.; Anderson, J. C.; Santoro, S. W.; Wang, L.; Martin, A. B.; King D. S.; Horn, D. M.; Schultz, P. G.
J. Am. Chem. Soc. 2003, 125, 935–939.
How Fast Is Polypeptide Biosynthesis?
Electron micrograph
• Polymerization
rate : 18 aa/sec
• 300 amino acid polypeptide in 20 sec in
E. coli;
• 100 amino acid polypeptide in 1 min. in
mammals.
• Protein synthesis represents ~30 % of ATP
utilized by E. coli.
http://arethusa.unh.edu/bchm752/ppthtml/april27/april27/sld025.htm.
Voet, D.; Voet, J. Biochemistry; Wiley: New York,1990.
Outline
¸ Protein Biosynthesis
¸ Unnatural Amino Acid Incorporation
¸ Chemical Acylation
¸ Exploiting Cell Machinery
¸ Using Existing Genetic Code
¸ First Unnatural Organism
¸ Future Directions
Outline
¸ Protein Biosynthesis
¸ Unnatural Amino Acid Incorporation
¸ Chemical Acylation
¸ Exploiting Cell Machinery
¸ Using Existing Genetic Code
¸ First Unnatural Organism
¸ Future Directions
The Central Dogma of Molecular Biology
Replication
DNA
messenger RNA
(mRNA)
transfer RNA
(tRNA)
ribosomal RNA
(rRNA)
Transcription
RNA
Protein
Translation
Voet, D.; Voet, J. Biochemistry; Wiley: New York,1990.
20 natural
L-amino acids
O
5'
-O P O
O3'
Base
O
OH H
DNA vs. RNA
DNA
RNA
O
5'
-O P O
O3'
Base
O
2'
OH OH
NH2
N
NH2
N
N
H
Adenine
N
N
H
N
N
Guanine
Guanine
N
NH2
NH2
N
N
Cytosine
N
H
O
O
Cytosine
O
O
NH
N
H
N
N
H
NH2
NH2
N
H
NH
O
NH
N
H
Adenine
N
O
N
N
O
NH
Thymine
N
H
O
Uracil
Main Elements For Protein Biosynthesis
• mRNA: template.
• Ribosome: polymerase.
• tRNACGAAla : amino acid-charged tRNA:
• tRNA
•Amino acid
•RS: aminoacyl-tRNA synthetase,
specific for each amino acid and capable
of charging most of tRNAs specific for
the same amino acid.
Lewin, B. Genes VII; Oxford: New York, Oxford University Press, 2000.
Transfer RNA (tRNA)
Acceptor or amino acid stem. A
7 bp stem that includes 5’-terminal
nucleotide.
All tRNAs terminate with
the sequence CCA with a
free 3’-OH group.
Anticodon arm. A 5 bp stem
ending in the loop that contains
the anticodon.
Voet, D.; Voet, J. Biochemistry; Wiley: New York,1990.
Codon–Anticodon Interaction
tRNAUACVal
anticodon
3'
mRNA
5'
C
A
U
G
U
A
• The proper tRNA is selected only through codon–anticodon
interactions
5'
3'
Charged Transfer RNA ( aa-tRNA)
H O
R C C _
NH3 O
+
+
ATP
H O
O
R C C O P O Ribose Adenine + PPi
_
NH3
O
+
• Charging
of tRNA is performed by
aminoacyl-tRNA synthetase (aa-RS).
•Many of aa-tRNA synthetases have
proofreading function
Aminoacyl-adenylate
(Aminoacyl-AMP)
Isoleucyl-tRNA synthetase:
1 Val / 50,000 Ile
tRNA
NH2
N
tRNA
O
O P _O
O
N
O
3' 2'
O OH
O C
H C R
NH3
+
Aminoacyl-tRNA
N
N
CH3 O
H3C
O
NH3
+
Isoleucine
_
CH3 O
H3C
O
NH3
+
Valine
Voet, D.; Voet, J. Biochemistry; Wiley: New York,1990.
_
Mechanism of Polypeptide Synthesis
OH
tRNA(n-1)
Exit
site
NH
Rn-1 CH
O C
NH
Rn CH
O C
NH
Rn-1 CH
O C
NH
Rn CH
O C
O
NH2
Rn+1 CH
O C
O
tRNA(n)
Peptidyl-tRNA
site
tRNA(n+1)
Acceptor
site
OH
tRNA(n)
Exit
site
NH
Rn+1 CH
O C
O
NH2
Rn+2 CH
O C
O
tRNA(n+1)
tRNA(n+2)
Peptidyl-tRNA
site
Acceptor
site
Voet, D.; Voet, J. Biochemistry; Wiley: New York,1990.
Outline
¸ Protein Biosynthesis
¸ Unnatural Amino Acid Incorporation
¸ Chemical Acylation
¸ Exploiting Cell Machinery
¸ Using Existing Genetic Code
¸ First Unnatural Organism
¸ Future Directions
Chemical Acylation of tRNA
5'
(amino acid)-AC–C
UAC
ATG
5'
3'
5'
(amino acid)-AC-PO3H
HO–C
5'
UAC
ATG
3'
Heckler, T. G.; Chang, L. -H.; Zama, Y.; Naka, T.; Chorghade, M. S.; Hecht. S. M. Biochemistry,
1984, 23, 1468–1473.
Chemical Acylation of tRNA
R
(NVOC)HN
H3CO
R
ClCH2CN
COOH
NEt3
CN
O
NO2
O
H3CO
DMF
Et3N
Cl
O
6-nitroveratrylcarbonyl chloride
(NVOC-Cl)
N
O
N
O
N
N
O
_
O
O
P
O
N
N
O
N
OH
OH
O
_
O
O P _O
O
O
5'-phospho-2-deoxycytidylyl(3',5')adenosine
pdCpA
O
_
O P O
_
O
N
N
N
N
N
pdCpA
H2N
H2N
H2N
H2N
O
(NVOC)HN
_
O
P
O
O
O
O
R
NH(NVOC)
O
O
OH
Robertson, S. A.; Ellman, J. A.; Schultz, P. G. J. Am. Chem. Soc. 1991, 113, 2722–2729.
Chemical Acylation of tRNA
H2N
H2N
N
O
N
N
N
N
O
O
_
O P O
_
O
5'
N
O
_
O
P
O
O
O
R
NH(NVOC)
O
O
OH
HO–C
T7
5'
3'
tRNA
gene
T7 RNA
polymerase
T4 RNA
ligase
UAC
5'
(aa)-AC–C
UAC
Bain, J. D.; Wacker, D. A.; Kuo, E. E.; Lyttle, M. H.; Chamberlin, A. R. J. Org. Chem, 1991, 56, 4615–4625.
Cload, S. T.; Liu, D. R.; Froland, W. A.; Schultz, P. G. Chem. Biol. 1996, 3, 1033–1038.
Unnatural Amino Acid Incorporation
5'
(aa)-ACC
5'
UAG
3'
In vitro
translation
Protein
containing unnatural
amino acid
mRNA
5'
• Possible
AUC
UAG
3'
multiple incorporation of one unnatural amino acid
• mRNA is unstable
• Need stoichiometric amounts of aminoacylated-tRNA.
Bain, J. D.; Wacker, D. A.; Kuo, E. E.; Lyttle, M. H.; Chamberlin, A. R. J. Org. Chem, 1991, 56, 4615–4625.
Cload, S. T.; Liu, D. R.; Froland, W. A.; Schultz, P. G. Chem. Biol. 1996, 3, 1033–1038.
Outline
¸ Protein Biosynthesis
¸ Unnatural Amino Acid Incorporation
¸ Chemical Acylation
¸ Exploiting Cell Machinery
¸ Using Existing Genetic Code
¸ First Unnatural Organism
¸ Future directions
Finding Unique Codon
Three STOP codons
do not encode an
amino acid.
UAA – ochre
UAG – amber
UGA – opal
Lewin, B. Genes VII; Oxford: New York, Oxford University Press, 2000.
Amber Codon Suppression
5'
(aa)-ACC
In vitro / in vivo
translation
5'
AUC
UAG
Protein
containing unnatural
amino acid
3'
Requirements for amber suppression :
• tRNACUA and RS must be orthogonal toE. coli system
• The resulting tRNACUAaa must recognize amber codon at the same rate
as in normal protein synthesis
Selecting For Orthogonal tRNACUA
TAG
ApR
S. cerevisiae
Gln-RNA
synthetase
Grown in the
presence of
ampicillin
sc-tRNACUAGln
survivors encode a suppressor
tRNA capable of inserting Gln
in response to the TAG codon
IC50 500 mg mL-1
E. coli
TAG
ApR
Grown in the
presence of
ampicillin
no growth observed
sc-tRNACUAGln
IC50 20 mg mL-1
E. coli
Liu, D. R.; Schultz, P. G. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 4780–4785.
Verifying the Orthogonality of GlnRS
S. cerevisiae
Gln-RNA
synthetase
Genomic GlnRS
deletion
no growth observed
E. coli
E. coli
Gln-RNA
synthetase
Genomic GlnRS
deletion
cells growth
E. coli
Liu, D. R.; Schultz, P. G. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 4780–4785.
Other tRNACUA/RS Orthogonal Pairs
Table 1. IC50 values (mg/mL) for control experiments
E. coli without suppressor tRNACUA
E. coli with supF expression
9.7
1700
Table 2. IC50 values (mg/mL) of screening experiments
tRNA source
tRNACUA
Saccharomyces
cerevisae tRNATyr
234
Homo sapiens
tRNATyr
1206
tRNACUA/aaRS
Saccharomyces
cerevisiae tRNAGln
20
500
Methanococcus
jannaschii tRNATyr
12
400
Liu, D. R.; Schultz, P. G. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 4780–4785.
Wang, L.; Magliery, T. J.; Liu, D. R.; Schultz, P. G. J. Am. Chem. Soc. 2000, 122, 5010–5011
Wang, L.; Schultz, P. G. Chem. Biol. 2001, 8, 883–890.
Engineering a Synthetase With Unnatural Amino
Acid Specificity
Engineering the
specificity of synthetase
Rational design
Combinatorial
approach
Difficult due to
high fidelity of
natural synthetase
Generate synthetase
mutant library
Select on their
specificity for
unnatural amino acid
Engineering a Synthetase With Unnatural Amino
Acid Specificity
TAG
M. jannaschii
Tyr-RS
mutant
library
ApR
orthogonal
mj-tRNACUATyr
add unnatural
amino acid
positive
selection/screen
survivors encode synthetases
that charge the orthogonal
suppressor tRNA with natural
or unnatural amino acid
E. coli
Wang, L.; Schultz, P. G. Chem. Biol. 2001, 8, 883–890.
Engineering a Synthetase With Unnatural Amino
Acid Specificity
survivors encode synthetases
that charge the orthogonal
suppressor tRNA with natural
or unnatural amino acid
TAG
barnase
survivors encode synthetase
that charge the orthogonal
suppressor tRNA with
unnatural amino acid only
No unnatural
Amino acid added
negative
selection/screening
synthetase
from positive
selection
E. coli
Wang, L.; Schultz, P. G. Chem. Biol. 2001, 8, 883–890.
orthogonal
mj-tRNACUATyr
Engineering a Synthetase With Unnatural Amino
Acid Specificity
TAG
M. jannaschii
Tyr-RS
mutant
library
ApR
orthogonal
mj-tRNACUATyr
add unnatural
amino acid
positive
selection/screen
survivors encode synthetases
that charge the orthogonal
suppressor tRNA with natural
or unnatural amino acid
E. coli
mutagenesis,
DNA shuffling
survivors encode synthetase
that charge the orthogonal
suppressor tRNA with
unnatural amino acid only
TAG
barnase
No unnatural
Amino acid added
negative
selection/screening
synthetase
from positive
selection
E. coli
Wang, L.; Schultz, P. G. Chem. Biol. 2001, 8, 883–890.
orthogonal
mj-tRNACUATyr
Outline
¸ Protein Biosynthesis
¸ Unnatural Amino Acid Incorporation
¸ Chemical Acylation
¸ Exploiting Cell Machinery
¸ Using Existing Genetic Code
¸ First Unnatural Organism
¸ Future directions
One Step Towards First “Unnatural” Organism
TAG
dihydrofolate
reductase
mj-Tyr-RNA
synthetase
In vivo
translation
SDS-PAGE gel and
Western blot analysis
mj-tRNATyr
CUA
E. coli
O
OH
H3CO
NH2
mj-tRNACUATyr
+
+
+
–
+
mutTyrRS
–
+
+
+
–
O-met-L-Tyr
–
+
–
+
+
wtTyrRS
+
–
–
–
–
O-methyl-L-tyrosine
Wang, L.; Brock, A.; Herberich, B.; Schultz, P. G. Science, 2001, 292, 498–500.
Synthesis of p-aminophenylalanine (p-AF)
O
HO
HO
OH
O
OH
O
OH
OH
O
OH
O
O
OH
OH
OH
O
OH
O
PapA
OH
O
O
NH2
NH2
O
Glucose
4-amino-4-deoxychorismate
chorismic
acid
O
PapB
4-amino-4deoxyprephenic
acid
PapC
NH2
O
OH
Proteins Papa, PapB and PapC
convert chorismate to paminophenylpyruvate
O
OH
aminotransferase
O
NH 2
p-aminophenylalanine
NH2
p-aminophenylpyruvate
Mehl, R. A.; Anderson, J. C.; Santoro, S. W.; Wang, L.; Martin, A. B.; King D. S.; Horn, D. M.; Schultz, P. G.
J. Am. Chem. Soc. 2003, 125, 935–939.
The First “Unnatural” Organism
sperm whale
myoglobin
TAG
Plpp
mjTyr-RNA
synthetase
papA
papB
papC
In vivo
translation
SDS-PAGE gel and
Western blot analysis
mjtRNATyr
CUA
E. coli
Mehl, R. A.; Anderson, J. C.; Santoro, S. W.; Wang, L.; Martin, A. B.; King D. S.; Horn, D. M.; Schultz, P. G.
J. Am. Chem. Soc. 2003, 125, 935–939.
The First “Unnatural” Organism
wt-tRNATyr
+
–
–
–
–
–
–
TyrRS
+
–
–
–
–
–
–
pAFRS
–
–
+
–
+
+
–
mutRNACUATyr
–
+
+
+
+
+
+
pAF
–
–
+
+
–
–
–
Plpp papA,papB,papC
–
–
–
–
–
+
+
2
3
4
5
6
7
8
Protein yield
Wild-type: 3.5 mg/L
Mutant:
3.0 mg/L
1
For the first time bacterium has:
• the ability to synthesize pAF from simple carbon sources;
• an aa-tRNA synthetase that uniquely utilizes pAF and no other
amino acid;
• a tRNA that is acylated by this synthetase and no other;
• the same tRNA delivers pAF efficiently into proteins in response to
the amber codon, TAG
Mehl, R. A.; Anderson, J. C.; Santoro, S. W.; Wang, L.; Martin, A. B.; King D. S.; Horn, D. M.; Schultz, P. G.
J. Am. Chem. Soc. 2003, 125, 935–939.
Outline
¸ Protein Biosynthesis
¸ Unnatural Amino Acid Incorporation
¸ Chemical Acylation
¸ Exploiting Cell Machinery
¸ Using Existing Genetic Code
¸ First Unnatural Organism
¸ Future Directions
Wobble Property of tRNA
“Wobble” is the property that allows
one anticodon to pair with two or three
codons
tRNAVal
anticodon
3'
mRNA
C
A
U
G
U
A
5'
5'
3'
or
mRNA
5'
G
U
G
3'
Table 3. Allowed wobble pairing
combinations in the third
codon–anticodon position
5’-anticodon base
3’-codon base
C
G
A
U
U
A or G
G
U or C
I
U, C or A
Voet, D.; Voet, J. Biochemistry; Wiley: New York,1990.
The Genetic Code
Lewin, B. Genes VII; Oxford: New York, Oxford University Press, 2000.
Using Wobble Property
L-proline is encoded by UUC and UUU
One tRNA with GAA anticodon recognizes both codons
Generation of tRNA with AAA anticodon might
recognize UUU codon with higher efficiency
Murine dihydrofolate reductase (mDHFR)
gene contains four UUC and five UUU codons
mDHFR had five substituted proline
residues with L-3-(2-naphthyl)alanine
O
H2N
OH
L-3-(2-naphthyl)alanine
Kwon, I.; Kirshenbaum, K.; Tirrell, D. A. J. Am. Chem. Soc. 2003, 125, 7512–7513
The Genetic Code
3 nt code gives 64 possible codons
4 nt code would give 256 codons
5 nt code would give 1024 codons
Lewin, B. Genes VII; Oxford: New York, Oxford University Press, 2000.
4 and 5 nt Codons
–CGC UAU GUA CUU ACC GGC CGU UAU GAC–
Arg Tyr Val Leu Thr Gly Arg Tyr Asp
Mutation at
Thr position
–CGC UAU GUA CUU AGGU GGC CGU UAU GAC–
Arg Tyr Val Leu Xaa Gly Arg Tyr Asp
Hohsaka, T.; Ashizuka, Y.; Sasaki, H.; Murakami, H.; Sisido, M. J. Am. Chem. Soc. 1999, 121, 12194–12195.
Magliery, T. J.; Anderson, J. C.; Schultz, P. G. J. Mol. Biol. 2001, 307, 755–769.
Increasing the Codon Size (4 nt codon)
tRNA
Codons
selected
CUUCCUAA
AGGA
CGCUAGGA
CUAG
AUUCUAAC
UAGA
CUGCCUAU
AGGC
GUAGGGUA
CCCU
IC50 500 mg mL-1
Suppression: 2.5–35 %
Magliery, T. J.; Anderson, J. C.; Schultz, P. G. J. Mol. Biol. 2001, 307, 755–769.
Increasing the Codon Size (5 nt codon)
Anticodon loop
Codon
Suppression (%)
CUGUCCUAA
AGGAC
5.0
CUGUCCUAA
AGGAU
11.3
CUAUUGGAC
CCAAU
4.4
UUGGUGGAA
CCACC
1.6
CUAGUGGAC
CCACU
7.4
GUGAUCCAA
CCAUC
8.0
UUGAUGGAG
CCAUC
5.6
CUGAGGGUC
CCCUC
3.8
UUGACCGAC
CGGUC
4.5
GUGGUAGGA
CUACC
7.4
UUAGUAGAU
CUACU
11.2
CUGAUAGAA
CUAGC
8.5
UUACUAGAC
CUAGU
12.0
Anderson, J. C.; Magliery, T. J.; Schultz P. G. Chem. Biol. 2002, 9, 237–244.
Novel Base Pairs
Requirements for the third base pair:
• stable and selective base pairing
• unnatural nucleoside must be membrane permeable
• must be stable inside the cell
• efficient and high fidelity enzymatic incorporation into DNA
N
N
7-aza-indole
Tae, E. L.; Wu, Y.; Xia G.; Schultz, P.G.; Romesberg, F. E. J. Am. Chem. Soc. 2001, 123, 7439–7440.
Summary and Conclusions
Chemical acylation:
• mRNA is not stable
• Need stoichiometric amount of charged tRNA
Exploiting cell machinery:
• orthogonal tRNA/RS pair is required
• engineered pair is capable to suppress amber
codon by inserting unnatural amino acid
Protein engineering:
• evolving better catalysts
• synthesizing proteins bearing D-amino acids
• synthesis of glycoproteins
OH
HO
HO
O
O
NHAc
NH2
OH
O
b-N-acetylglucosamine (GlcNAc)-L-serine
Zhang, Z.; Gildersleeve, J.; Yang, Y.; Xu, R.; Loo, J. A.; Uryu, S.; Wong, C., Schultz, P. G. Science
2004, 303, 371–373.
Acknowledgments
Prof. John Frost
Dr. Karen Frost
Frost Group Members
Dr. Jihane Achkar
Wei Niu
Jiantao Guo
Ningqing Ran
Xiaofei Jia
Heather Stueben
Man Kit Lau
Dr. Dongming Xie
Kin Sing Stephen Lee
Jinsong Yang
Wensheng Li
Dr. Jian Yi
Mapitso Molefe
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