1. SPECTROSCOPIC STUDIES OF GREEN FLUORECENCE PROTEIN (GFP)
AT HIGH PRESSURES
Seung-Joon Jeon1, Deok-Su Kim1, Hang-Sun Ahn1,
Mu Hyun Choi2, Hackjin Kim3
1Department of Chemistry and Center for Electro- & Photo-Responsive Molecules,
Korea University, Seoul , Korea
2School of Life Science and Biotechnology, Korea University, Seoul , Korea
3Department of Chemistry, Chungnam National University, Taejon , Korea

2. Green Fluorescent Protein(GFP)
Shimomura et al discovered as a companion protein to aequorin
from Aequrea jellyfish
Shimomura identified the chromophore for absorption & fluorescence
4-(p-hydroxybenzylidene)imidazolidin-5-one
Prasher et al cloning of the gene
Chalfie et al & Inouye et al
demonstrated that expression of the gene in other organism
creates fluorescence, which means that the gene contains all the
information necessary for posttranslational synthesis of the chromophore
very useful fluorescence marker in biological cells !
a lot of mutant GFP and various applications

3. 11-stranded β-barrel pH 5.5~12 stable
α-helices
238 amino acids
GFP MW= 27kDa
The diameter of about 24 Å and the length of about 42 Å
pH 5.5~12 stable
Neutral buffer stable up to 65℃

4. Chromorphore (4-(p-hydroxybenzylidene)imidazolidin-5-one)
For photochemistry and photophysics,
Interaction between the chromophore and its surrounding is important :
- protonation state of the chromophores and its surrounging
- hydrogen-bonding network around chromophore

5. Absorption and Fluorescence spectrum of GFP family

6. GFP(2-5) amino acids sequence
GFP(2-5) amino acid sequence (EGFP, S65T type)
Mutation : - improving folding
- fluorescence characteristics
- decreasing surface hydrophobicity
- decreasing thermosensitivity
GFP(2-5) amino acids sequence 1 2 3 4 5 6 7 8 9 60
val
thr
pht
tyr
gly
gla
cys
160
ile
lys
ala
asn
phe
arg
his
170
glu
asp
gln
leu

7. GFP(2-5) Absorbance and Fluorescence spectrum
Blue line: Absorbance
main peak:487nm
shoulder peak:458nm
Black line: Fluorescence
main peak:509nm
shoulder peak:532nm

8. High Pressure Experiments
GFP(2-5) : concentration .29mg/ml
High pressure generation
Piston-cylinder cell (lower P) DAC (higher P)
Liquid state range of solvent(water)
Buffer : pressure effect
Pressure unit : 1 atm ≈ 1bar = 0.1 MPa

9. Piston – Cylinder Cell System (up to 6500 atm & High T)
computer

10. DAC (diamond anvil cell) System(above 6500 atm)
Pressure gasket
holder
support

11. Liquid range of water with pressure and temperature
At room temperature
up to atm liquid
At high pressures (2000atm)
down to -20Co liquid
cold denaturation possible

12. Protonation and deprotonation
~390nm
~480nm

13. GFP Absorbance and Fluorescence spectrum on pH effect

14. Buffer-solution and high pressure
Phosphate buffer
Under High pressure
Phosphate buffer
(pressure effect + pH effect)
Tris-HCl buffer
Tris-HCl buffer

15. GFP Absorbance and Fluorescence spectrum under High pressure
pH=8 , Tris-HCl buffer solution
a : Fluorescence spectrum
b : Absorbance spectrum

16. High pressure effect
Absorbance and Fluorescence intensities are reduced with similar behavior
100atm~6000atm: reversible(80-90%)
Over 7.5kbar : irreversible
(a lot of volume reduction)
Assuming a two-state model
at high pressures, the fluorescence state transfers to the nonfluorescence state

17. ΔV0 calculate ΔV0= -14.25 ㎖/mol(1000-3500atm)
(Fluo. State)
(Nonfluo. State)
ΔV0= ㎖/mol( atm)
㎖/mol( atm)
(1000atm)

18. Reduction of absorption intensity
GFP has a large central cavity, which contain the chromophore
Prevent the chromophore rotational freedom, especially by a hula-twist and in a the φ angle

19. High pressure phase transition
Broader pressure range transition
reduction of β-can volume
Around 3500atm
aggregation
Around 7000atm
pressure-induced denature

20. Aggregation formation from
recovered GFP after pressurized
Aggregation

21. Under high pressure and different pH’s
pH=5.8~9.80 under high pressure pH
Transition pressure
pH=5.8
1000atm
pH=6.0~6.8
1500atm
pH=8.0~9
3500atm
Over pH=9.8
1200atm
pH=6.4 at 4250atm
Exponential decay
First order transfer dynamics

22. Phase Diagram of Protein
Actual shape, size, and orientation
of elliptic boundary are defined by
Six thermodynamic parameters,
Elliptic phase diagram of protein
By S.A. Hawley(1971)
Based on only two possible state
equlibrium thermodynamic
and reversible unfolding

23. Tongue like diagram
(chymotrypsinogen)
Hillside type diagram
(ribonuclease)

24. High Pressure and Temperature effects
Pressure : constant
Temperature: increase (1℃/min)
2500atm 75℃
100atm 75℃

25. Denatured
Native

26. Summary Pressure Induced Transition
up to 3500atm reduced absorption, volume reduction
up to 7000atm aggregation
above 7500 atm - fluorescence disappear & irreversible
Native sate
Unfolding sate
two steps
Pressure-Temperature effect
GFP : Tongue-like elliptic P-T diagram