GFP Protein

8/9/2019 GFP Protein

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GFP Protein and Variants:GFP Protein and Variants:

Why so many possible colors?Why so many possible colors?


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Everything should be made as simple as possible, but not simpler.

Albert Einstein


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Jellyfish?...and?...Jellyfish?...and?...

Aequorea victoria: a luminescent/fluorescent organism (as many)


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Green Fluorescent Protein (GFP)Green Fluorescent Protein (GFP)

Interlude:

Do you know the difference between luminescence andfluorescence/phosphorescence or incandescence?

Why marine organism emit light in the blue-green region of the electromagneticspectrum?

In A. victoria, GFP fluorescence occurs when luminescent aequorin proteininteracts with Ca2+ ions, inducing a blue glow.

Mechanism: some of the luminescent energy (Frster Resonance EnergyTransfer, FRETsee later for applications) is transferred to GFP, shifting theoverall color towards green.


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The diversity of genetic mutations is illustrated by this San Diego beach scenedrawn with living bacteria expressing 8 different colors of fluorescent proteins.

The chromatic spectra of GFP variantsThe chromatic spectra of GFP variants


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http://www.microscopyu.com/articles/livecellimaging/fpintro.html


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GFP: A secondary/tertiary structure analysisGFP: A secondary/tertiary structure analysis

-barrel protein

Chromophore


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Elastic model

Interlude:Why stretching your tendons or muscles is so difficult?What is the difference between stretching a metal wire or a tendon?


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RamachandranRamachandran plot:plot:

a simplified Potential Energy Surface (PES)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?


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RamachandranRamachandran plotplot


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Walking on Potential Energy SurfacesWalking on Potential Energy Surfaces

Walking on the red path instead of the green path makes a difference, though thefinal point is the same.

so proteins can be seen asso proteins can be seen as mechanicalmechanical systemssystems

(keep it is as a grant, for the moment)(keep it is as a grant, for the moment)


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Exploring theExploring the LysozymeLysozyme PES of foldingPES of folding


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The geometry of FoldingThe 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 oneof three stable conformations the failure can fold in 3198 different combinations


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Lets go back to GFP fluorescenceLets go back to GFP fluorescence

ChromophoreChromophore based on abased on a ImidazolinoneImidazolinone ringring


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Molecules can translate, rotate and vibrateMolecules can translate, rotate and vibrate

Vibrational modes of water

Rotational modes of water

or electronically excited between 2 electronic statesor electronically excited between 2 electronic states


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Definition of fluorescence and phosphorescenceDefinition of fluorescence and phosphorescence

Start to distribute electrons in two energy levels:we get 3 possible combinations

E

Interlude:By which spectroscopy do you check the different electronic states in amolecule?

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?


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The electromagnetic spectrumThe electromagnetic spectrumThermal luminescenceThermal luminescence

Interlude:Why, if the UV radiation has more energy does not heat while IR radiation yes?


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VibronicVibronic transitions (transitions (vibrationalvibrational + electronic)+ electronic)


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On this simple diagram you learn and deduce a lot!!!!!On this simple diagram you learn and deduce a lot!!!!!

Fluorescence and phosphorescenceFluorescence and phosphorescence

isc = intersystem crossingvr = vibrational relaxationic = internal conversionec = external collision

E


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Interlude:Can you explain the wavelength shift between absorption, fluorescenceand phosphorescence?The meaning of quencing?The meaning of bleaching?


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The FRET mechanismThe 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?...


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Previous InterludesPrevious Interludes

(now you can answer extensively(now you can answer extensively))

Do you know the difference between luminescence andfluorescence/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 radiationdoes?


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http://www.olympusconfocal.com/java/fpfluorophores/index.html

PostPost--translational maturation of GFPtranslational maturation of GFP chromophorechromophore


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a) Is the precursor sequence Thr-Tyr-Gly necessary to get the chromophore?

lets check the GFP variants

Some questions ariseSome questions arise..

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?


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b) let us suppose to introduce a sequence like Gly-Gly-Gly.

this is the result (a non-fluorescent chemical species)

NoNo aminoacidaminoacid sequence dependency!! The chemistry issequence dependency!! The chemistry is

containedcontained in the aminoacids but not the resultingin the aminoacids but not the resulting

fluorescencefluorescence


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NoNo aminoacidaminoacid sequence dependency!!sequence dependency!!

The chemistry isThe chemistry is containedcontained in all the aminoacids butin all the aminoacids but

not all compounds are fluorescentnot all compounds are fluorescent

However why so many colors with different variants?However why so many colors with different variants?


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Interlude:Can you propose why we need the xxx-Tyr-Gly sequence to get fluorescence?Something related to coniugation?...

Gly-Gly-Gly product

Thr-Tyr-Gly product


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How toHow to change the color in a dye?change the color in a dye?

Electron in a boxElectron in a box


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n = 1

n = 2

n = 3

n = 4

Electrons in a linear conjugated moleculeElectrons in a linear conjugated molecule

Interlude:What is the condition to get conjugation in molecules?


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Cu

Fe

e-

Electron into a circleElectron into a circle

Interlude: How do you imagine electrons in a ring? Do you recall some chemical

species?...


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How toHow to change the color in the GFPchange the color in the GFP

chromophorechromophore??

Interlude:Wait for your answer.


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However a question is still open:However a question is still open:

If there is no dependency from theIf there is no dependency from the aminoacidaminoacid sequence, whysequence, why

GFP is so specific to obtain the fluorophore?GFP is so specific to obtain the fluorophore?


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Lets check the GFP structureLets check the GFP structure


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F ti f th GFPFormation of the GFP h hchromophore i b dis based


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Formation of the GFPFormation of the GFP chromophorechromophore is basedis based

on aon a

mechanical stressmechanical stress

induced by theinduced by the

protein skeleton on the specific positionprotein skeleton on the specific position

where thewhere the chromophorechromophore will be synthesized!!will be synthesized!!


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Take Home MessageTake Home Message

By a mix of chemico-physical informations and

protein folding, we have been able to understandthe GFP mechanism of chromophore maturationand fluorescence conditions.

Nextenjoy the genetic engineering

courseand think atomistic!!

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GFP Protein