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 energye.g. UV light from sun causes the sunburn

not the red visible light

FluorescenceE

NE

RG

Y

S0

S1

S2

T2

T1ABS FL I.C.

ABS - Absorbance S 0.1.2 - Singlet Electronic Energy LevelsFL - Fluorescence T 1,2 - Corresponding Triplet StatesI.C.- Nonradiative Internal Conversion IsC - Intersystem Crossing PH - Phosphorescence

IsC

IsC

PH

[Vibrational sublevels]

Jablonski Diagram

Vibrational energy levelsRotational energy levelsElectronic energy levels

Singlet States Triplet States

fast slow (phosphorescence)Much longer wavelength (blue ex – red em)

Triplet state

Simplified Jablonski Diagram

S0

S’

1E

n er g

yS1

hvex hvem

FluorescenceStokes 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 nmFluoresceinmolecule

Flu

ores

cen

ce I

nte

nsit

y

Wavelength

Ethidium

PE

cis-Parinaric acid

Texas Red

PE-TR Conj.

PI

FITC

600 nm300 nm 500 nm 700 nm400 nm457350 514 610 632488 Common Laser Lines

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

FITC 488 525PE 488 575APC 630 650PerCP™ 488 680Cascade Blue 360 450Coumerin-phalloidin 350 450Texas Red™ 610 630Tetramethylrhodamine-amines 550 575CY3 (indotrimethinecyanines) 540 575CY5 (indopentamethinecyanines) 640 670

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 505 511

NBD Golgi 488 525

DPH Lipid 350 420

TMA-DPH Lipid 350 420

Rhodamine 123 Mitochondria 488 525

DiO Lipid 488 500

diI-Cn-(5) Lipid 550 565

diO-Cn-(3) Lipid 488 500

Probe Site Excitation Emission

BODIPY - borate-dipyrromethene complexes NBD - nitrobenzoxadiazoleDPH – diphenylhexatriene TMA - trimethylammonium

Fluorescence

Resonance Energy Transfer

Inte

nsi

ty

Wavelength

Absorbance

DONOR

Absorbance

Fluorescence Fluorescence

ACCEPTOR

Molecule 1 Molecule 2

FRET propertiesIsolated 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 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?