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 ENERGY S0S0 S1S1 S2S2 T2T2 T1T1 ABS FL I.C. ABS - AbsorbanceS 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

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

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

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

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 FITC PE APC PerCP ™ Cascade Blue Coumerin-phalloidin Texas Red ™ Tetramethylrhodamine-amines CY3 (indotrimethinecyanines) CY5 (indopentamethinecyanines) Probe Excitation Emission

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

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

Specific Organelle Probes BODIPY Golgi NBD Golgi DPH Lipid TMA-DPH Lipid Rhodamine 123 Mitochondria DiOLipid diI-Cn-(5)Lipid diO-Cn-(3)Lipid Probe Site Excitation Emission BODIPY - borate-dipyrromethene complexes NBD - nitrobenzoxadiazole DPH – diphenylhexatriene TMA - trimethylammonium

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

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)

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

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

Table 13.2

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

Aminoacyl-tRNA Synthetase

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.

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