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A Genetically Encoded Fluorescent Amino Acid Background for the Schultz paper in June ’06 PNAS PNAS Overview • • • • • What is fluorescence Use of fluorophores How can you make a molecule fluorescent Protein synthesis Protein folding 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 Fluorescence Jablonski Diagram Singlet States Triplet States Vibrational energy levels Rotational energy levels Electronic energy levels S2 ENERGY T2 S1 IsC T1 ABS FL fast S0 I.C. Triplet state PH IsC slow (phosphorescence) Much longer wavelength (blue ex – red em) [Vibrational sublevels] ABS - Absorbance S 0.1.2 - Singlet Electronic Energy Levels FL - Fluorescence T 1,2 - Corresponding Triplet States I.C.- Nonradiative Internal Conversion IsC - Intersystem Crossing PH - Phosphorescence Simplified Jablonski Diagram S’ 1 S1 hvex S0 hvem Fluorescence Stokes Shift Fluorescence Intensity – is the energy difference between the lowest energy peak of absorbance and the highest energy of emission Fluorescein molecule Stokes Shift is 25 nm 495 nm Wavelength 520 nm 350 300 nm 457 488 514 400 nm 500 nm Common Laser Lines 610 632 600 nm 700 nm PE-TR Conj. Texas Red PI Ethidium PE FITC cis-Parinaric acid Jellyfish genes • Why use GFP – abundant in organism – cloned – doesn’t need post-trans modifications – can expressed in many diff organisms – good marker protein – fluorescent 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 Probes for Proteins Probe FITC PE APC PerCP™ Cascade Blue Coumerin-phalloidin Texas Red™ Tetramethylrhodamine-amines CY3 (indotrimethinecyanines) CY5 (indopentamethinecyanines) Excitation 488 488 630 488 360 350 610 550 540 640 Emission 525 575 650 680 450 450 630 575 575 670 Microarray Immuno-Phenotyping (labeled antibody) TLC (plate matrix is fluor) Fluorescent Microscope Arc Lamp EPI-Illumination Excitation Diaphragm Excitation Filter Ocular Dichroic Filter Objective Emission Filter Specific Organelle Probes Probe BODIPY NBD DPH TMA-DPH Rhodamine 123 DiO diI-Cn-(5) diO-Cn-(3) Site Golgi Golgi Lipid Lipid Excitation 505 488 350 350 Mitochondria 488 Lipid 488 Lipid 550 Lipid 488 BODIPY - borate-dipyrromethene complexes DPH – diphenylhexatriene Emission 511 525 420 420 525 500 565 500 NBD - nitrobenzoxadiazole TMA - trimethylammonium Fluorescence Resonance Energy Transfer Molecule 1 Molecule 2 Fluorescence Fluorescence ACCEPTOR DONOR Absorbance Absorbance Wavelength FRET properties Isolated donor Donor distance too great Donor distance correct 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) Eng ptn w/ GFP Synth peptide w/ NBD-aa Dye (FM464) Protein Synthesis • Stages • Components • How can the system be altered to incorporate unnatural amino acids Table 13.2 Amber suppressor 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. Aminoacyl-tRNA Synthetase An expanding genetic code T. Ashton Croppa and Peter G. Schultzb, 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. Protein folding, Unfolding, and Refolding Why is folding important?