Background for the Schultz paper in
June ’06 PNAS

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

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

S
2
Singlet States
Jablonski Diagram
Triplet States
Vibrational energy levels
Rotational energy levels
Electronic energy levels
T
2
S
1
ABS
IsC
FL fast
I.C.
PH
T
1
Triplet state
IsC slow (phosphorescence)
Much longer wavelength ( blue ex
– red em)
[Vibrational sublevels]
S
0
ABS - Absorbance
FL - Fluorescence
S 0.1.2 - Singlet Electronic Energy Levels
T 1,2 - Corresponding Triplet States
I.C.- Nonradiative Internal Conversion IsC - Intersystem Crossing PH - Phosphorescence

Simplified Jablonski Diagram
1
S’
S
1 hv ex hv em
S
0

Stokes Shift
– 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 520 nm
Wavelength

350
300 nm 400 nm
457 488 514 610 632
500 nm 600 nm 700 nm
Common Laser Lines
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

• 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

Probe
FITC
PE
APC
PerCP ™
Cascade Blue
Coumerin-phalloidin
Texas Red ™
Tetramethylrhodamine-amines
CY3 (indotrimethinecyanines)
CY5 (indopentamethinecyanines)
Excitation Emission
488
488
630
488
360
350
610
550
540
640
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

Probe Site Excitation Emission
BODIPY
NBD
Golgi
Golgi
505
488
DPH
TMA-DPH
Lipid
Lipid
350
350
Rhodamine 123 Mitochondria 488
DiO Lipid 488 diI-Cn-(5) diO-Cn-(3)
Lipid
Lipid
550
488
511
525
420
420
525
500
565
500
BODIPY - borate-dipyrromethene complexes NBD - nitrobenzoxadiazole
DPH – diphenylhexatriene TMA - trimethylammonium

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

Isolated donor
Donor distance too great
Donor distance correct

• Chemically add
– Not always specific
– Perturbing
– Direct vs Indirect
• Synthetically incorporate
– Limited to small molecules
• Biosynthetically incorporate
– Genetically engineer
– GFP and derivatives large (>20kD)

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

• 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.

An expanding genetic code
T. Ashton Cropp a and Peter G. Schultz 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?