jessica torres

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

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

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

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

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

The Genetic Alphabet: 20 Common Amino Acids

Expanding The Genetic Alphabet: Unnatural Amino Acids

An Orthogonal Biosynthetic Machinery

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.

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

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

pyrrolysine

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

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.

Genetically Encoded Alkenes for the Expression of Protein with New Function

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.

Bioconjugation with Alkene Lysine via the Thiol-ene Reaction

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.

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

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

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

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

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

Bioconjugation with ‘Turn-on’ Fluorescence

Non-fluorescent Fluorescent

9

Nature Chem. 2012, 4, 298-304

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

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.

Acknowledgements

Deiters Lab Dr. Alexander Deiters Dr. Chungjung (Hank) Chou

Collaborators: Jason Chin Lab (MRC, Cambridge, UK) Kathrin Lang, Lloyd Davis

NSF Graduate Fellowship

Duke University

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