1. Genetic Incorporation of Unnatural Amino Acids into Proteins Monica Amin Yang Song Yan Liu Harbani Malik Vipul Madahar Jiayu Liao Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, New York Department of Bioengineering, University of California, Riverside

2. GOALS Protein Azide-alkyne Huisgen cycloaddition reaction: Specific binding, no change in conformation of protein Use an unnatural amino acid as a chemical handle for site- specific immobilization Current Method Protein Our Method Different reaction sites on the protein

3. SUMOylation Pathway Several proteins catalyze covalent conjugation between SUMO and cellular target proteins that are involved in regulation of various cellular processes. Disregulation of the pathway is linked to diseases like ovarian carcinoma, melanoma, and lung adenocarcinoma 3. Cascade event involving multiple protein-protein interactions SUMO: Small – Ubiquitin – like MOdifiers Figure 1. Yang Song

4. FRET – Based Analysis Binding assays based on steady state and time resolved FRET can be used to monitor interactions in the SUMOylation Pathway Forster (Fluorescence) Resonance Energy Transfer Non- radiative process Donor (Cypet) and Acceptor (Ypet) (Fluorophores) Donor transfers energy to ground state acceptor Proximity of 1-10nm Dipole- dipole interactions Figure 2. FRET principle. Angewandte Chemie (2006)

5. Site- Specific Incorporation of the unnatural amino acid UAG Site-specific incorporation of unnatural amino acid, ppropargyloxy- phenylalanine (pPpa) [Figure 3], into Cypet-SUMO1 in Escherichia coli. Mutated M. Janaschii tyrosyl-tRNA synthetase created to selectively charge an amber suppressor tRNA with pPpa. Figure 3. Nature Methods (2007 ) Figure 4. ChemCommun (2002)

6. Site- Specific Incorporation of the Unnatural Amino Acid Once we have our DNA construct with the TAG mutation DNA gets transcribed to mRNA [TAG  UAG] In response to this unique codon the tRNA with the unnatural amino acid attaches to the mRNA After the translation, the unnatural amino acid is incorporated into the peptide sequence PCR Mutation SUMO SerHisTag Start Codon: ATG Stop Codon: TAA SUMO TAGHisTag Start Codon: ATG Stop Codon: TAA

7. Immobilization on Glass Plate Azide-Alkyne Huisgen Cycloaddition Achieve site-specific immobilization of a fluorescent tag protein (Cypet- SUMO1) on azide modified glass surface under mild conditions Detect the resonance energy transfer with Ypet tagged enzymes in SUMOylation pathway, like ubc9, AOS1/Ubo2, SENPs, and PIASs. Figure 5. Bioorganic & Medicinal Chemistry Letters (2005)

8. SUMO1 gene- commercial plasmid PCR amplify SUMO1 and TAG SUMO1 (specifically designed primers) Ligation of SUMO1 and TAG-SUMO1 using TOPO cloning vector, pCR2.0 Transformation using TOP 10 cells DNA Extraction Characterization: Digestion Check, Sequencing cDNA Cloning METHODS : CLONING A+ pCR2.0- TOPO (3.9kb) K+ Cloning Region

9. Gene Cloning Digestion of SUMO1-pCR2.0 and pET-28B vector (specific digestion enzymes) Ligation of SUMO1 gene to pET-28B vector Transformation using TOP10 cells DNA Extraction & Sequencing Clone TAG- SUMO1 into SUMO1- pET-28B vector [1] Clone Cypet gene into TAG-SUMO1- pET-28B vector [2] METHODS : CLONING TAG-HisSUMO1Cypet NcoINotINdeI 2 1 pET- 28B (5368bp) Restriction Sites for : 1. TAG-SUMO  NotI, NcoI 2. TAG-SUMO-Cypet  NotI, NdeI

10. METHODS: PROTEIN EXPRESSION & PURIFICATION Protein Expression: 1. Transform TAG-Cypet- SUMO1 plasmid, orthogonal tRNA and tRNA synthetase plasmids into BL21 cells 2. Grow Transformed cells (step 1) in presence of unnatural amino acid and related antibiotics in the medium Protein Purification: Use column chromatography (Nickel-NTA Agarose column) and dialysis FRET based Protein- Protein Interaction: Determine the interaction between TAG-Cypet-SUMO1 and Ypet- Ubc9 and compare to no mutation interaction

11. RESULTS A 1 2 3 4 5 A 6 7 8 9 10 (a) (b) Figure (Right) Digestion gel of the TAG- SUMO1/pET-28B plasmid. (a)TAG-pET-28B ~5kbp (b)SUMO1 ~300bp 1-8, 10 were positive and well 9 was negative.

12. RESULTS Incorporated Cypet fluorescence gene using cloning procedures mentioned in methods We grew the cells on a Kanamycin resistant agar plate; got colonies Sent for sequencing.

13. Proof of Concept We determined the interaction between Cypet-SUMO1 and Ypet- Ubc9 using FRET [Figure on next slide]. Cypet-SUMO1 is excited at 414nm Emission from Cypet- SUMO1 slowly decreases as the absorption of Ypet- Ubc9 gradually increases do to the increasing concentration of Ypet- Ubc9. Cypet- SUMO1Ypet-Ubc9Dialysis Buffer 5 uL0 uL15 uL 5 uL1 uL14 uL 5 uL2 uL13 uL 5 uL3 uL12 uL Kept constant Cypet- SUMO1 concentration and gradually increased Ypet- Ubc9 concentration Denotes the specific interaction between SUMO1 and Ubc9.

14. RFU Emission Wavelength in nm Cypet-SUMO1 and Ypet- Ubc9 Proof of Concept

15. Amber stop codon –TAG has been successfully incorporated into SUMO1/pET-28B plasmid to recognize unnatural amino acid. TAG incorporated Cypet-SUMO1/pET-28B construct is currently being studied. The TAG- Cypet-SUMO1/pET-28B will allow us to site- specifically incorporate pPpa into interested proteins. SUMMARY

16. FUTURE DIRECTIONS Use FRET- based assays to monitor protein-protein interaction within the SUMOylation Pathway Use protein micro array [protein immobilization on glass plate] to find the inhibitors in the Sumoylation pathway Incorporate unnatural amino acids in proteins in the mammalian system

17. Acknowledgments THANK YOU Jun Wang Dr. Rodgers BRITE Program Dr. Liao Dr. Liao’s Lab National Science Foundation

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