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CHEM 1004 – Interfaces

Chemistry and Biology – GFP


Introduction

• GFP is a protein that fluoresces green and is isolated from the jellyfish Aequorea
victoria

• It is unique as it only requires oxygen to activate the activity, and does not
require other enzymes (or biomolecules)

• This allows GFP to be used to tag cells – if an organism expresses GFP, it will
fluoresce

Discovery

• Shimomura isolated a bioluminescent protein (aequorin) that gave off blue light
(when in contact with Ca2+ in seawater) but the jellyfish glowed green. This was
due to a second jellyfish protein (GFP) which in turn re-emitted green light

• The ability of GFP to process blue light to green (its fluorescence) was found to
be integral to its structure, occurring without the need for any accompanying
factors

• Chalfie succeeded in introducing the gene for GFP into the DNA of a small worm
using molecular biological techniques. GFP was produced by the cells, giving off
its green glow without the need for addition of any extra components, and without
any indication of causing damage to the worms (no chemical intervention)

• How cells become green


Chalfie positioned the GFP-gene behind a promoter (a gene switch), which is active in the touch receptor neurons of
the round worm. He injected the gene construct into the gonads of a mature worm. The worm is a hermaphrodite and
fertilizes itself. The GFP gene is passed onto the eggs that the worm lays. The eggs divide, forming new individuals.
The GFP-gene is then present in all cells of the new generation of roundworms, but only the touch receptor neurons
will produce GFP. When they fill up with GFP, they start to glow green under ultraviolet light.
• Tsien studied how GFP’s structure produces the observed green fluorescence,
and then used this knowledge to tweak the structure to produce molecules that
emit light at slightly different wavelengths, which gave tags of different colours.
(i.e. Tsien worked out the mechanism)

• Mutations of the protein allows the chromophore to be modified. It keeps the


same action but with a different colour, intensity and lifetime

Structure

• Two 11-stranded β barrels and α helix down the centre of the barrel containing
tripeptide unit (serine-tyrosine-glycine) which is the source of the chromophore

• But it is not the actual chromophore as it becomes active during reaction with
molecular oxygen
(unfolded / denatured / floppy conformation → folding → cyclization → dehydration → aerial
oxidation to give the rigid and conjugated π system (resonance) of the chromophore)

Usesα

• Fluorescence Resonance Energy Transfer (FRET) spectroscopy can be used to


measure distances using GFP to enable tracking of molecular processes

• Molecules can transfer energy without giving off radiation (but need to be in close
proximity)