This document discusses incorporating unnatural amino acids into proteins to expand their functions. It describes how pyrrolysine is used as an orthogonal system to incorporate unnatural amino acids specified by an amber stop codon. Various unnatural amino acids containing alkene and norbornene functional groups were successfully incorporated into GFP and other proteins in E. coli and mammalian cells. These modified proteins could then be site-specifically labeled using bioorthogonal reactions like the thiol-ene and Diels-Alder reactions. This allows proteins to be labeled and studied in live cells with new functions.

1. Incorporation of Unnatural Amino Acids
for the Expression of Proteins with New
Function
Jessica Torres-Kolbus, Chungjung Chou, Kathrin Lang, Lloyd
Davis, Jason Chin, and Alexander Deiters*
University of Puerto Rico, Cayey
RISE Seminar
September 20, 2012

2. Proteins: Many Structures, Many Functions
Nature Chemical Biology 1, 13 - 21 (2005)
• Compose over 50% of the cellular dry mass
• Involved in all cellular functions:
• Signaling
• Transport
• Defense
• Catalysis
• Maintenance
• Stability
• Tens of thousands of different proteins
Green Fluorescent Protein (GFP)
Bright green fluorescence
Aequorea victoria
238 AAs
Myoglobin (Mb)
Binds to iron and oxigen
Found in muscle tissue of most vertebrates
154 AAs

3. Why Study Proteins?
• understand protein structure-function relationships
• investigate protein-involved biological processes
• many diseases are caused by errors in proteins, e.g.:
cystic fibrosis – one amino acid deletion
sickle cell anemia – one incorrect amino acid position
Huntington disease – expansion repeat of an amino acid
• manipulate proteins, protein-based drugs, generate proteins and organisms with
new properties

4. How Study Proteins?
• many proteins undergo post-translational modifications or bind to cofactors to extend their
properties
• biological processes are very complex and are regulated in both space and time
• many of these processes cannot be observed and studied when the protein involved is
isolated
• study of biological processes in their native environments
• reporter tags are required for the trafficking and detection of biomolecules

5. Strategies for Chemical Modification of Proteins
Protein
Labeling
Labeled proteins (e.g. fluorescent tags) provide exciting new tools for studying proteins and
their function in the cell

6. The Genetic Alphabet: 20 Common Amino Acids

7. Expanding The Genetic Alphabet: Unnatural Amino Acids

8. An Orthogonal Biosynthetic Machinery

9. Stealing Parts from other Organisms
→ large differences between archaea,
bacteria, and eukaryotes in tRNA genes
and their aminoacyl tRNA synthetases
→ engineer a synthetase to specifically
recognize an UAA
PylRS is found in some methanogenic archea and
bacteria, charges its cognate tRNA with pyrrolysine.
The unique and large substrate binding pocket of
pyrrolysyl synthetase (PylRS) shows that it may
recognize a broad spectrum of lysine UAA.
Pyrrolysyl synthetase (PylRS)
binding pocket
Yokoyama, S. Acta Crystallogr Sect F Struct Biol Cryst Commun 2006, 62 (Pt 10), 1031-3.

10. A Pyrrolysyl-Based Facile System
The PylRS/tRNA pair is orthogonal in E. coli, S. cerevisiae , mammalian cells, and C. elegans.
Pyrrolysine is naturally encoded by an amber stop codon

11. Promiscuity of the PylRS
Effective Pyl mimics: Ineffective Pyl mimics:
pyrrolysine

12. Promiscuity of the PylRS
Effective Pyl mimics: Ineffective Pyl mimics:

13. Unnatural Amino Acids as Bioorthogonal Chemical Reporters
Bertozzi, C. R. Nat. Chem. Bio. 2005, 1 (1): 13–21.
1. Site-specific incorporation of UAA with reactive handle.
2. Highly selective reaction with exogenously delivered probe.
Bioorthogonal reaction refers to any reaction that takes place inside of biological systems
with selective reactivity and biocompatibility.
 Physiological conditions
 No-cross reaction
 Non-toxic
 Low concentrations
 High yields
 Fast
The reaction results in a stable covalent bond between the protein and the probe.

14. Genetically Encoded Alkenes for the Expression
of Protein with New Function

15. Introduction of the Alkene Functionality into Proteins
• Rarely found in natural proteins.
• Versatile in organic transformations.
Alloc-L-lysine is incorporated
via the wild-type PylRS.

16. Bioconjugation with Alkene Lysine via the Thiol-ene Reaction

17. Incorporation Efficiency of Alkene Lysine Library
100% (AllocLys) 80% 109%
82% 86% 14% 9%
3.8% 102% 45%
Each amino acid was tested as PylRS substrate for incorporation into protein.
Incorporation efficiency relative to AllocLys.

18. Expression of Alkene-modified sfGFP
100% (AllocLys) 10 80% 109%
82% 3 86% 4 14% 9%
3.8% 102% 45%
Each amino acid was tested as PylRS substrate for incorporation into protein.
Incorporation efficiency relative to AllocLys.
58
30
46
M -AA WT +10 +3 +4
25
Incorporation in sfGFP in E. coli

19. Fluorescent labeling of sfGFP
Fluorescence
Comassie
Light activated, site-specific labeling of sfGFP bearing an alkene
with a thiol-containing fluorescent probe via the thiol-ene reaction
Samples were irradiated at 365 nm for 5 min
10
3
Dansyl-thiol (fluorescent)Alkene-modified
protein

20. Bioconjugation of sfGFP and lysozyme
M
58
30
46
25
1 2 3 4 5 6 7 8
M. Marker
1. wt sfGFP
2. +10, - lysozyme
3. wt sfGFP + lysozyme, - UV
4. +10 + lysozyme, - UV
5. +3 + lysozyme, - UV
6. wt sfGFP + lysozyme, + UV
7. +10 + lysozyme, + UV
8. +3 + lysozyme, + UV
sfGFP increased from ~28 kDa to ~44 kDa after conjugating to the lysozyme with UV light
Samples were irradiated at 365 nm for 5 min

21. Diels-Alder reaction.
• High selectivity
• High yields
• Fast reaction in aqueous media
Synthesis of Norbornene Lysine and Protein Expression
Nature Chem. 2012, 4, 298-304

22. Diels-Alder reaction.
• High selectivity
• High yields
• Fast reaction in aqueous media
Synthesis of Norbornene Lysine and Protein Expression
Nature Chem. 2012, 4, 298-304
Expression in E. coli

23. Bioconjugation with ‘Turn-on’ Fluorescence
Non-fluorescent Fluorescent
9
Nature Chem. 2012, 4, 298-304

24. Site-Specific Protein Labeling via Bioorthogonal Cycloaddition with Genetically
Encoded Norbornene in Mammalian Cells
Labeling of EGFR-(TAG)-GFP in HEK293 cells.
Nature Chem. 2012 , 4, 298-304
TAMRA

25. Summary and Conclusions
• Site-specific incorporation of UAAs into proteins in both bacteria and
mammalian cells.
• Labeled proteins via bioorthogonal reactions.
• A library of aliphatic alkene lysines was generated for genetic encoding into
proteins.
• The alkene-modified protein was successfully subjected to bioorthogonal
labeling via the thiol-ene reaction.
• A norbornene-containing amino acid was synthesized and encoded into
protein for fast ‘turn-on’ fluorescence labeling in both bacterial and
mammalian cells.

26. Acknowledgements
Deiters Lab
Dr. Alexander Deiters
Dr. Chungjung (Hank) Chou
Collaborators:
Jason Chin Lab (MRC, Cambridge, UK)
Kathrin Lang, Lloyd Davis
NSF Graduate Fellowship

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