GFP Protein and Variants:
Why so many possible colors?
Everything should be made as simple as possible, but not simpler.
Albert Einstein
Jellyfish?...and?...
Aequorea victoria: a luminescent/fluorescent organism (as many…)
Green Fluorescent Protein (GFP)
In A. victoria, GFP fluorescence occurs when luminescent aequorin protein
interacts with Ca2+ ions, inducing a blue glow.
Mechanism: some of the luminescent energy (Förster Resonance Energy
Transfer, FRET…see later for applications…) is transferred to GFP, shifting the
overall color towards green.
Interlude:
Do you know the difference between “luminescence” and
fluorescence/phosphorescence or incandescence?
Why marine organism emit light in the blue-green region of the electromagnetic
spectrum?
The chromatic spectra of GFP variants
The diversity of genetic mutations is illustrated by this San Diego beach scene
drawn with living bacteria expressing 8 different colors of fluorescent proteins.
http://www.microscopyu.com/articles/livecellimaging/fpintro.html
GFP: A secondary/tertiary structure analysis
Chromophore
β-barrel protein
“Elastic model”
Interlude:
Why “stretching” your tendons or muscles is so difficult?
What is the difference between stretching a metal wire or a tendon?
Ramachandran plot:
a simplified Potential Energy Surface (PES)
The assumption is easy. Based on the:
a) hydrogen bond connections;
b) on the dihedral angles;
we can determine, uniquely, the secondary structure in a protein.
Interlude:
Why the aforementioned assumptions are good?
Can you define a PES?
Ramachandran plot
Walking on Potential Energy Surfaces
Walking on the red path instead of the green path makes a difference, though the
final point is the same.
…so proteins can be seen as “mechanical” systems…
(keep it is as a grant, for the moment)
Exploring the Lysozyme PES of folding
The geometry of Folding
Interlude:
Do you see here the solution of the “Levinthal paradox”?....
For example a polypeptide 100 residues long will have 99 peptide bonds and so
198 different phi and psi bond angles. If each of these bond angles can be in one
of three stable conformations the failure can fold in 3198 different combinations
Lets go back to GFP fluorescence
Chromophore based on a Imidazolinone ring
Molecules can translate, rotate and vibrate
Vibrational modes of water
Rotational modes of water
or electronically excited between 2 electronic states
Definition of fluorescence and phosphorescence
Start to distribute electrons in two energy levels:
we get 3 possible combinations
Interlude:
By which “spectroscopy” do you check the different electronic states in a
molecule?
Do you know the difference in energy (roughly) between electronic, vibrational,
rotational and translational levels in a molecules?
How do you define the “electron spin”?
The electromagnetic spectrum
Thermal luminescence
Interlude:
Why, if the UV radiation has more energy does not “heat” while IR radiation yes?
Vibronic transitions (vibrational + electronic)
Fluorescence and phosphorescence
isc = intersystem crossing
vr = vibrational relaxation
ic = internal conversion
ec = external collision
On this simple diagram you learn and deduce a lot!!!!!
Interlude:
Can you explain the wavelength shift between absorption, fluorescence
and phosphorescence?
The meaning of quencing?
The meaning of bleaching?
The FRET mechanism
Depending from:
Overlap of the emission-adsorption spectrum of the emitter-absorber distance
…CFP = cyan Fluorescent Protein, YFP = yellow fluorescent protein, how can be?...
Previous Interludes
(now you can answer extensively…)
Do you know the difference between “luminescence” and
fluorescence/phosphorescence or incandescence?
Why marine organism emit light at the blue-green region of the electromagnetic
spectrum?
Why, if the UV radiation has more energy does not “heat” while IR radiation
does?
Post-translational maturation of GFP chromophore
http://www.olympusconfocal.com/java/fpfluorophores/index.html
Some questions arise….
a) Is the precursor sequence Thr-Tyr-Gly necessary to get the chromophore?
…lets check the GFP variants…
Ser65-Tyr66-Gly67 = EGFP
Gln66-Tyr67-Gly68 = DsRed
Lys66-Tyr67-Gly68 = ZsYellow
Met63-Tyr64-Gly65 = far-red fluorescent protein, eqFP611
Glu64-Tyr65-Gly66 = HcRed
His62-Tyr63-Gly64 = Kikume Green-Red
Interlude:
The Tyr-Gly sequence is a must. Why?...what experiment do you propose?
b) …let us suppose to introduce a sequence like Gly-Gly-Gly….
…this is the result (a non-fluorescent chemical species)
No aminoacid sequence dependency!! The chemistry is
“contained” in the aminoacids but not the resulting
fluorescence
No aminoacid sequence dependency!!
The chemistry is “contained” in all the aminoacids but
not all compounds are fluorescent
However why so many colors with different variants?
Thr-Tyr-Gly product
Gly-Gly-Gly product
Interlude:
Can you propose why we need the xxx-Tyr-Gly sequence to get fluorescence?
Something related to “coniugation”?...
“How to” change the color in a dye?
Electron in a box
Electrons in a linear conjugated molecule
n=4
n=3
n=2
n=1
Interlude:
What is the condition to get conjugation in molecules?
Electron into a circle
e-
Fe
Cu
Interlude:
How do you imagine electrons in a ring? Do you recall some chemical
species?...
“How to” change the color in the GFP
chromophore?
Interlude:
Wait for your answer….
However a question is still open:
If there is no dependency from the aminoacid sequence, why
GFP is so specific to obtain the fluorophore?
Lets check the GFP structure
Formation of the GFP chromophore is based
on a “mechanical stress” induced by the
protein skeleton on the specific position
where the chromophore will be synthesized!!
Take Home Message
By a mix of chemico-physical informations and
protein folding, we have been able to understand
the GFP mechanism of chromophore maturation
and fluorescence conditions.
Next…enjoy the genetic engineering
course…and think atomistic!!
