synthesis of proteins containing unnatural amino acids

Synthesis Of Proteins ContainingUnnatural Amino Acids

Michigan State UniversityDepartment of Chemistry

Justas Jancauskas

Why Introduce Unnatural Amino Acids?

• Drug industry: N-methylated proteins are stable toproteolysis

• Selective protein labeling with antigenic orfluorophoric 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?

• Polymerization rate : 18 aa/sec• 300 amino acid polypeptide in 20 sec inE. coli;• 100 amino acid polypeptide in 1 min. inmammals.

• Protein synthesis represents ~30 % of ATPutilized by E. coli.

Electron micrograph

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

The Central Dogma of Molecular Biology

DNA

RNA Protein

Replication

Transcription

Translationribosomal RNA (rRNA)

transfer RNA (tRNA)

messenger RNA (mRNA)

20 naturalL-amino acids

Voet, D.; Voet, J. Biochemistry; Wiley: New York,1990.

RNA

DNA vs. RNADNA

N

NNH

NNH2

NH

NNH

NO

NH2

N

NH

NH2

O

NH

NH

O

O

Adenine

Guanine

Cytosine

Thymine

Base

O

HOH

OP-OO-

O

3'

5' Base

O

OHOH

OP-OO-

O

NH

NH

O

OUracil

N

NNH

NNH2

NH

NNH

NO

NH2

N

NH

NH2

O

Adenine

Guanine

Cytosine

2'3'

5'

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 capableof charging most of tRNAs specific forthe same amino acid.

Lewin, B. Genes VII; Oxford: New York, Oxford University Press, 2000.

Transfer RNA (tRNA)

Anticodon arm. A 5 bp stemending in the loop that containsthe anticodon.

Acceptor or amino acid stem. A7 bp stem that includes 5’-terminalnucleotide.

All tRNAs terminate withthe sequence CCA with afree 3’-OH group.


Codon–Anticodon Interaction

• The proper tRNA is selected only through codon–anticodoninteractions

G U AmRNA

5' 3'

C A U

tRNAUACVal

anticodon

3' 5'

Charged Transfer RNA ( aa-tRNA)• Charging of tRNA is performed byaminoacyl-tRNA synthetase (aa-RS).

•Many of aa-tRNA synthetases haveproofreading function

R CH

NH3

C

+O

O_ + ATP

R CH

NH3

C

+

OO P

O

OO Ribose Adenine PPi_

Aminoacyl-adenylate(Aminoacyl-AMP)

N

NN

NNH2

O

OHO

OPOO

O_

tRNA

CNH3

H R

+

tRNA

Aminoacyl-tRNA

3' 2'

+

CO

Isoleucyl-tRNA synthetase:1 Val / 50,000 Ile

H3CCH3

NH3+

O

O_

Isoleucine

H3C O

CH3 O

NH3+

_

Valine


Mechanism of Polypeptide Synthesis


NHCHRn-1CONHCHRnCOO

tRNA(n)

NH2CHRn+1COO

tRNA(n+1)

Peptidyl-tRNAsite

Acceptorsite

Exitsite

OH

tRNA(n-1)

OH

tRNA(n)

NHCHRn-1CONHCHRnCO

O

tRNA(n+1)

NHCHRn+1CO

NH2CHRn+2COO

tRNA(n+2)

Peptidyl-tRNAsite

Acceptorsite

Exitsite

Outline





Chemical Acylation of tRNA

ATG5' 3'UAC

5'(amino acid)-AC–C

ATG5' 3'UAC

5'(amino acid)-AC-PO3H HO–C

Heckler, T. G.; Chang, L. -H.; Zama, Y.; Naka, T.; Chorghade, M. S.; Hecht. S. M. Biochemistry,1984, 23, 1468–1473.


Robertson, S. A.; Ellman, J. A.; Schultz, P. G. J. Am. Chem. Soc. 1991, 113, 2722–2729.

(NVOC)HN COOH

R(NVOC)HN O CN

R

O

N

N

H2N O

O

OP OOO

__

O PO

O

O

_ O

N

NN

N

H2N

OH

NH(NVOC)

O O

R

ClCH2CN

NEt3

pdCpADMFEt3N

H3CO

H3CO

NO2

O Cl

O6-nitroveratrylcarbonyl chloride

(NVOC-Cl)

N

N

H2N O

O

OP OOO

O PO

O

O

O

N

NN

N

H2N

OH

pdCpA5'-phospho-2-deoxycytidylyl(3',5')adenosine

_

__

OH


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.

T75' 3'

T7 RNApolymerase

T4 RNAligasetRNA

gene UAC

5'HO–C

UAC

5'(aa)-AC–C

N

N

H2N O

O

OP OOO

__

O PO

O

O

_ O

N

NN

N

H2N

OH

NH(NVOC)

O O

R

Unnatural Amino Acid Incorporation

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.

• Possible multiple incorporation of one unnatural amino acid

• mRNA is unstable

• Need stoichiometric amounts of aminoacylated-tRNA.

UAG5' 3'AUC

5'(aa)-ACC

Proteincontaining unnatural

amino acid

In vitrotranslation

UAG5' 3'

mRNA

Outline




¸ Future directions

Finding Unique Codon

Three STOP codonsdo not encode anamino acid.

UAA – ochreUAG – amberUGA – opal


Amber Codon Suppression

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

UAG5' 3'AUC

5'(aa)-ACC

Proteincontaining unnatural

amino acid

In vitro / in vivotranslation

E. coli

Selecting For Orthogonal tRNACUA

S. cerevisiae Gln-RNA

synthetase

TAGApR

IC50 500 mg mL-1

IC50 20 mg mL-1

sc-tRNACUAGln

Grown in the presence of ampicillin survivors encode a suppressor

tRNA capable of inserting Glnin response to the TAG codon

E. coli

TAGApR

sc-tRNACUAGln

Grown in the presence of ampicillin

no growth observed

Liu, D. R.; Schultz, P. G. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 4780–4785.

Verifying the Orthogonality of GlnRS

S. cerevisiaeGln-RNA

synthetaseno growth observed

E. coliGln-RNA

synthetasecells growth

Liu, D. R.; Schultz, P. G. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 4780–4785.

Genomic GlnRSdeletion

E. coli

Genomic GlnRSdeletion

E. coli

Other tRNACUA/RS Orthogonal Pairs

40012Methanococcusjannaschii tRNATyr

50020Saccharomycescerevisiae tRNAGln

1206Homo sapienstRNATyr

234Saccharomycescerevisae tRNATyr

tRNACUA/aaRStRNACUAtRNA sourceTable 2. IC50 values (mg/mL) of screening experiments

1700E. coli with supF expression9.7E. coli without suppressor tRNACUA

Table 1. IC50 values (mg/mL) for control experiments

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, 5010–5011Wang, L.; Schultz, P. G. Chem. Biol. 2001, 8, 883–890.

Engineering a Synthetase With Unnatural AminoAcid Specificity

Engineering the specificity of synthetase

Rational design Combinatorial approach

Generate synthetasemutant library

Select on their specificity for

unnatural amino acid

Difficult due tohigh fidelity of

natural synthetase

E. coli


positiveselection/screen

M. jannaschii Tyr-RS mutant

library

TAGApR

orthogonalmj-tRNACUA

Tyr

add unnaturalamino acid survivors encode synthetases

that charge the orthogonalsuppressor tRNA with naturalor unnatural amino acid

Wang, L.; Schultz, P. G. Chem. Biol. 2001, 8, 883–890.


survivors encode synthetasesthat charge the orthogonalsuppressor tRNA with naturalor unnatural amino acid

barnase

synthetasefrom positive

selectionnegative selection/screening

TAG


Tyr

E. coli

survivors encode synthetasethat charge the orthogonalsuppressor tRNA withunnatural amino acid only

No unnaturalAmino acid added



barnase

synthetasefrom positive

selection

TAG


Tyr

survivors encode synthetasesthat charge the orthogonalsuppressor tRNA with naturalor unnatural amino acid

E. coli

negative selection/screening

survivors encode synthetasethat charge the orthogonalsuppressor tRNA withunnatural amino acid only

No unnaturalAmino acid added

E. coli

M. jannaschii Tyr-RS mutant

library

TAGApR


Tyr

mutagenesis,DNA shuffling

positiveselection/screen

add unnaturalamino acid


Outline




¸ Future directions

One Step Towards First “Unnatural” Organism

SDS-PAGE gel and Western blot analysis

In vivotranslation

Wang, L.; Brock, A.; Herberich, B.; Schultz, P. G. Science, 2001, 292, 498–500.

E. coli

mj-tRNATyrCUA

mj-Tyr-RNAsynthetase

TAGdihydrofolate

reductase

–+–+

–––+wtTyrRS+–+–O-met-L-Tyr+++–mutTyrRS–+++mj-tRNACUA

Tyr

OH

O

NH2H3CO

O-methyl-L-tyrosine

Synthesis of p-aminophenylalanine (p-AF)

O OH

OHOH

HO

HO

GlucoseOH

OHO

NH2

OHO

NH2

PapA PapB

PapC

chorismicacid

4-amino-4-deoxychorismate 4-amino-4-deoxyprephenic acid

p-aminophenylpyruvate

O OHO

O NH2

OHO

O OHO

O

OHO

O

OHO

O

Proteins Papa, PapB and PapCconvert chorismate to p-aminophenylpyruvate

NH2

aminotransferase

p-aminophenylalanine

OH

O

NH2

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

SDS-PAGE gel and Western blot analysis

In vivotranslation

mjtRNATyrCUA

mjTyr-RNAsynthetase

TAGsperm whale

myoglobin

Plpp

papApapB

papC

E. coli


The First “Unnatural” Organism

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 otheramino acid;• a tRNA that is acylated by this synthetase and no other;• the same tRNA delivers pAF efficiently into proteins in response tothe amber codon, TAG

Protein yieldWild-type: 3.5 mg/LMutant: 3.0 mg/L

––––––+wt-tRNATyr

++–––––Plpp papA,papB,papC–––++––pAF++++++–mutRNACUA

Tyr

–++–+––pAFRS––––––+TyrRS

87654321


Outline





Wobble Property of tRNA

G U AmRNA

5' 3'

C A U

tRNAVal anticodon

3' 5'

G U GmRNA

5' 3'

or

“Wobble” is the property that allowsone anticodon to pair with two or threecodons

U, C or AIU or CGA or GU

UAGC

3’-codon base5’-anticodon base

Table 3. Allowed wobble pairingcombinations in the thirdcodon–anticodon position


The Genetic Code


Using Wobble Property

L-proline is encoded by UUC and UUUOne tRNA with GAA anticodon recognizes both codons

Generation of tRNA with AAA anticodon mightrecognize UUU codon with higher efficiencyMurine dihydrofolate reductase (mDHFR)

gene contains four UUC and five UUU codons

Kwon, I.; Kirshenbaum, K.; Tirrell, D. A. J. Am. Chem. Soc. 2003, 125, 7512–7513

mDHFR had five substituted proline residues with L-3-(2-naphthyl)alanine

OH

O

L-3-(2-naphthyl)alanine

H2N

The Genetic Code

3 nt code gives 64 possible codons4 nt code would give 256 codons5 nt code would give 1024 codons


4 and 5 nt Codons

–CGC UAU GUA CUU ACC GGC CGU UAU GAC– Arg Tyr Val Leu Thr 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.

–CGC UAU GUA CUU AGGU GGC CGU UAU GAC– Arg Tyr Val Leu Xaa Gly Arg Tyr Asp

Mutation atThr position

Increasing the Codon Size (4 nt codon)

Magliery, T. J.; Anderson, J. C.; Schultz, P. G. J. Mol. Biol. 2001, 307, 755–769.

CCCUGUAGGGUAAGGCCUGCCUAUUAGAAUUCUAACCUAGCGCUAGGAAGGACUUCCUAA

CodonsselectedtRNA

IC50 500 mg mL-1

Suppression: 2.5–35 %

Increasing the Codon Size (5 nt codon)

UUACUAGACCUGAUAGAAUUAGUAGAUGUGGUAGGAUUGACCGACCUGAGGGUCUUGAUGGAGGUGAUCCAACUAGUGGACUUGGUGGAACUAUUGGACCUGUCCUAACUGUCCUAAAnticodon loop

7.4CUACC11.2CUACU8.5CUAGC

12.0CUAGU

3.8CCCUC4.5CGGUC

1.6CCACC7.4CCACU8.0CCAUC5.6CCAUC

4.4CCAAU11.3AGGAU5.0AGGAC

Suppression (%)Codon

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

Tae, E. L.; Wu, Y.; Xia G.; Schultz, P.G.; Romesberg, F. E. J. Am. Chem. Soc. 2001, 123, 7439–7440.

NN

7-aza-indole

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 ambercodon by inserting unnatural amino acid

Protein engineering:• evolving better catalysts• synthesizing proteins bearing D-amino acids• synthesis of glycoproteins

OHOHO

NHAcO

OH

OH

O

NH2

b-N-acetylglucosamine (GlcNAc)-L-serine

Zhang, Z.; Gildersleeve, J.; Yang, Y.; Xu, R.; Loo, J. A.; Uryu, S.; Wong, C., Schultz, P. G. Science2004, 303, 371–373.

Acknowledgments

Frost Group Members

Dr. Jihane AchkarJiantao GuoXiaofei JiaMan Kit LauKin Sing Stephen LeeWensheng LiMapitso Molefe

Wei NiuNingqing RanHeather StuebenDr. Dongming XieJinsong YangDr. Jian Yi

Prof. John FrostDr. Karen Frost

synthesis of proteins containing unnatural amino acids

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