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 136-701, Korea 2School of Life Science and Biotechnology, Korea University, Seoul 136-701, Korea 3Department of Chemistry, Chungnam National University, Taejon 305-764, Korea

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

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℃

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

Absorption and Fluorescence spectrum of GFP family

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

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

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

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

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

Liquid range of water with pressure and temperature At room temperature up to 10000 atm liquid At high pressures (2000atm) down to -20Co liquid cold denaturation possible

Protonation and deprotonation ~390nm ~480nm

GFP Absorbance and Fluorescence spectrum on pH effect

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

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

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

ΔV0 calculate ΔV0= -14.25 ㎖/mol(1000-3500atm) (Fluo. State) (Nonfluo. State) ΔV0= -14.25 ㎖/mol(1000-3500atm) -23.73 ㎖/mol(3500-5500atm) (1000atm)

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

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

Aggregation formation from recovered GFP after pressurized Aggregation

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

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

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

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

Denatured Native

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