1. A Genetically Encoded Fluorescent Amino Acid Background for the Schultz paper in June ’06 PNAS

2. PNAS

4. Overview What is fluorescence Use of fluorophores How can you make a molecule fluorescent Protein synthesis Protein folding

5. Fluorescence The longer the wavelength the lower the energy The shorter the wavelength the higher the energy e.g. UV light from sun causes the sunburn not the red visible light

6. Fluorescence ENERGY S0S0 S1S1 S2S2 T2T2 T1T1 ABS FL I.C. ABS - AbsorbanceS 0.1.2 - Singlet Electronic Energy Levels FL - FluorescenceT 1,2 - Corresponding Triplet States I.C.- Nonradiative Internal ConversionIsC - Intersystem CrossingPH - Phosphorescence IsC PH [Vibrational sublevels] Jablonski Diagram Vibrational energy levels Rotational energy levels Electronic energy levels Singlet StatesTriplet States fast slow (phosphorescence) Much longer wavelength (blue ex – red em) Triplet state

7. Simplified Jablonski Diagram S0S0 S’ 1 Energy S1S1 hv ex hv em

9. Fluorescence Stokes Shift –is the energy difference between the lowest energy peak of absorbance and the highest energy of emission 495 nm 520 nm Stokes Shift is 25 nm Fluorescein molecule Fluorescence Intensity Wavelength

11. Ethidium PE cis-Parinaric acid Texas Red PE-TR Conj. PI FITC 600 nm300 nm500 nm700 nm400 nm 457350514610632488 Common Laser Lines

13. Uses for fluorescent probes in biology Tracking –Qualitative Imaging –in vitro –in vivo –Quantitative DNA, protein, lipids, ions, signaling molecules –Relative amts, absolute amts, environment, interactions Nearly as sensitive as radioactivity, and a lot safer

14. Probes for Proteins FITC488525 PE488575 APC630650 PerCP ™ 488680 Cascade Blue360450 Coumerin-phalloidin350450 Texas Red ™ 610630 Tetramethylrhodamine-amines 550575 CY3 (indotrimethinecyanines) 540575 CY5 (indopentamethinecyanines) 640670 Probe Excitation Emission

15. TLC (plate matrix is fluor) Immuno-Phenotyping (labeled antibody) Microarray

16. Fluorescent Microscope Dichroic Filter Objective Arc Lamp Emission Filter Excitation Diaphragm Ocular Excitation Filter EPI-Illumination

18. Specific Organelle Probes BODIPY Golgi505511 NBD Golgi488525 DPH Lipid350420 TMA-DPH Lipid350420 Rhodamine 123 Mitochondria 488525 DiOLipid488500 diI-Cn-(5)Lipid550565 diO-Cn-(3)Lipid488500 Probe Site Excitation Emission BODIPY - borate-dipyrromethene complexes NBD - nitrobenzoxadiazole DPH – diphenylhexatriene TMA - trimethylammonium

20. Fluorescence Resonance Energy Transfer Intensity Wavelength Absorbance DONOR Absorbance Fluorescence ACCEPTOR Molecule 1Molecule 2

21. FRET properties Isolated donor Donor distance too great Donor distance correct

22. How can I label MFM? Chemically add –Not always specific –Perturbing –Direct vs Indirect Synthetically incorporate –Limited to small molecules Biosynthetically incorporate –Genetically engineer –GFP and derivatives large (>20kD)

23. Dye (FM464) Synth peptide w/ NBD-aa Eng ptn w/ GFP

24. Protein Synthesis Stages Components How can the system be altered to incorporate unnatural amino acids

27. Table 13.2

28. A mutant allele coding for a tRNA whose anticodon is altered in such a way that the suppressor tRNA inserts an amino acid at an amber codon in translation suppressing (preventing) termination. Amber suppressor

29. Aminoacyl-tRNA Synthetase

30. An expanding genetic code T. Ashton Cropp a and Peter G. Schultz b, a b More than 30 novel amino acids have been genetically encoded in response to unique triplet and quadruplet codons including fluorescent, photoreactive and redox active amino acids, glycosylated and heavy atom derived amino acids in addition to those with keto, azido and acetylenic chains. In this article, we describe recent advances that make it possible to add new building blocks systematically to the genetic codes of bacteria, yeast and mammalian cells. Taken together these tools will enable the detailed investigation of protein structure and function, which is not possible with conventional mutagenesis. Moreover, by lifting the constraints of the existing 20-amino-acid code, it should be possible to generate proteins and perhaps entire organisms with new or enhanced properties.

33. Protein folding, Unfolding, and Refolding Why is folding important?