29
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

Jessica torres

Embed Size (px)

Citation preview

Page 1: 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

Page 2: Jessica torres

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

Page 3: Jessica torres

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

Page 4: Jessica torres

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

Page 5: Jessica torres

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

Page 6: Jessica torres

The Genetic Alphabet: 20 Common Amino Acids

Page 7: Jessica torres

Expanding The Genetic Alphabet: Unnatural Amino Acids

Page 8: Jessica torres

An Orthogonal Biosynthetic Machinery

Page 9: Jessica torres

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.

Page 10: Jessica torres

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

Page 11: Jessica torres

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

pyrrolysine

Page 12: Jessica torres

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

Page 13: Jessica torres

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.

Page 14: Jessica torres

Genetically Encoded Alkenes for the Expression of Protein with New Function

Page 15: Jessica torres

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.

Page 16: Jessica torres

Bioconjugation with Alkene Lysine via the Thiol-ene Reaction

Page 17: Jessica torres

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.

Page 18: Jessica torres

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

Page 19: Jessica torres

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

Page 20: Jessica torres

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

Page 21: Jessica torres

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

Page 22: Jessica torres

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

Page 23: Jessica torres

Bioconjugation with ‘Turn-on’ Fluorescence

Non-fluorescent Fluorescent

9

Nature Chem. 2012, 4, 298-304

Page 24: Jessica torres

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

Page 25: Jessica torres

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.

Page 26: Jessica torres

Acknowledgements

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

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

NSF Graduate Fellowship

Page 27: Jessica torres

Duke University

UNC-Chapel Hill

NCSU RTP

•  largest university in the state, with almost 30,000 students

•  more than 1,700 faculty members, many of them nationally recognized

•  offers master's degrees in 101 fields, and doctoral degrees in 59 fields, in addition to the Doctor of

Veterinary Medicine degree

•  has 10 colleges and schools: Colleges of Agriculture and Life Sciences, Design, Education, Engineering,

Natural Resources, Humanities and Social Sciences, Management, Physical and Mathematical Sciences,

Textiles, and Veterinary Medicine

•  located west of downtown Raleigh, a part of the state’s technology-rich Research Triangle area

North Carolina State University

http://www.ncsu.edu/ http://www.rtp.org/ http://www.unc.edu/index.htm http://www.duke.edu/

Page 28: Jessica torres

Department of Chemistry at NCSU

Dabney Hall Main Campus

http://www.ncsu.edu/chemistry/

•  Over 120 graduate students and 27 research faculty

•  20 to 40 new graduate students each year

•  Analytical, Biological Chemistry, ChemEd, Inorganic, Magnetic resonance,

Materials, Nanoscience, Organic, Physical, Polymers, Theoretical

Page 29: Jessica torres

Join the Department of Chemistry at NCSU

•  Bachelor’s degree in Chemistry or related fields

•  GPA of at least 3.0 (out of 4.0) in the sciences

•  GRE General Test scores are required and the Subject Test is recommended

•  Application – opens online Aug 15, closes Dec 15

http://www.ncsu.edu/grad/applygrad.htm

•  Statement of Purpose

•  One official transcript

•  Three reference letters

•  No application fee

http://www.ncsu.edu/chemistry/graduate/application.html