J. RúlR aúl GiGrigera · Free energy of denaturation ΔG P 0 Cold denaturation Heat denaturation...
Transcript of J. RúlR aúl GiGrigera · Free energy of denaturation ΔG P 0 Cold denaturation Heat denaturation...
The role of water in the d t ti f t i ddenaturation of proteins under
high pressurehigh pressure(Or how to cook an egg at room temperature)
J R úl G iJ. Raúl GrigeraIFLYSIBIFLYSIB
(University of La Plata and CONICET, Argentina)
Free energy of denaturation
ΔG.
P0
Heat denaturationCold denaturation
0
22 78
5/6/2010 Denaturation by pressure 2T/C‐100 ‐22 78
0 100
T / °C T / °CT / C T / C
5/6/2010 Denaturation by pressure 3
T / ˚C
Free energy of denaturation
ΔG.
P0P1 < P0 P2 > P0
0
5/6/2010 Denaturation by pressure 4T/C‐100 0 100
From Clausius‐ Clapeyron equation
Integrating between T‐T0 y P ‐P0
with
p
5/6/2010 Denaturation by pressure 55/6/2010Cold denaturation
5
The previous expression for p pΔG assume that:
= Const
Which is not trueWhich is not true. Taking the next term
= Const
we get different shape
= Constshape
Accepting that condition we get:5/6/2010 Denaturation by pressure 65/6/2010
Cold denaturation 6
Accepting that condition we get:
5/6/2010 Denaturation by pressure 7
Under that conditions the PT diagram is elliptic.
pPressure denaturation
Colddenaturation
T
denaturationNative
5/6/2010 Denaturation by pressure 85/6/2010Cold denaturation
8
Heat denaturation
4000p/bar
3000
2000 Chymotrypsinogen (pH 2.07)
1000
0 10 20 30 40 500
5/6/2010 Denaturation by pressure 95/6/2010Cold denaturation
9
0 0 0 30 40 50
T /°CHawley S.A. Biochemistry, 10, 2436 (1971).
4000
/b4000
3000
p/bar
3000
p/bar
2000
Ribonuclease (pH 2)3000
Ribonuclease (pH 2)20002000
10001000
00
5/6/2010 Denaturation by pressure 105/6/2010Cold denaturation
10
0 10 20 30 40 50T /°C
5/6/2010Cold denaturation
10
0 10 20 30 40 500
T /°C
We will focus on the hydrophobic i iinteraction
• The solubility of non‐polar solutes decreasesThe solubility of non polar solutes decreases with temperature, therefore:
ΔΔ) solsolsolSTHTG Δ−Δ=Δ )(
)()( 12 TGTG Δ>Δfor T1 < T2
• Then we have
0ΔS 0<Δ solS
5/6/2010 Denaturation by pressure 11
Ka t man propose that the decreasing inKautzman propose that the decreasing in entropy is due to the hydrophobic hydration
ΔS < 0Hydrationsphere ΔS < 0p
5/6/2010Desnaturalización en frío
AFA ‐ Rosario 2009 12
Goldammer, Hertz. J. Phys. Chem (1970) NMRHallenga, Grigera JR, Berendsen. J. Phys. Chem (1980) Relaj. Dielect..Rezus, Bakker Phys Rev, Letters (2007) IR
5/6/2010Desnaturalización en frío
AFA ‐ Rosario 2009 13
, y , ( )
Hydrophobic Interactionyd op ob c te act o
ΔSIh>0
ΔSHh<0
Approx 1kcal/mol (4 kJ/mol)
5/6/2010Desnaturalización en frío
AFA ‐ Rosario 2009 14
.
• The hydrophobic hydration, and consequently the hydrophobic interaction, q y y pcan be produced due to the capacity of water to form hydrogen bond networkswater to form hydrogen bond networks.
• The HB network, remain under anythermodynamics condition?thermodynamics condition?
5/6/2010 Denaturation by pressure 15
η/η1barViscosity
20.36 oC1.00
6.24 oC
0.98
4.00 oC0.96
2 25 oC
0.96
0 94 2.25 C0.94
p / kbar
0.920.0 0.3 0.6 0.9 1.2 1.5
5/6/2010 Denaturation by pressure 16
p / kbar
Horne R.A, Johnson D.S. J. Phys Chem. 70, 2182‐2190 (1966)
Translational(D) and rotational (1/τ2) diffusion coefficientscoefficients
2.5
D 1/τ2 ar
)
-30 oC2.0
2
/ x(1
b 30 C
x(p)
/
0 oC
1.5
0 oC
90 oC1.0
0 1 2 3p / kbar
5/6/2010 Denaturation by pressure 17
p / kbar•Wolf. L.A. J. Chem. Soc. Faraday Trans. I. 71,784‐79 (1975)
.
E i t l id h th t th• Experimental evidences show that the pressure produce a weakening on the hydrogen bond networking of water phenomenon that start innetworking of water , phenomenon that start in the range of 1‐2 kbar. Around that range the distinctive characteristics of water change their gtrend.
• How can we define the crossover ?
5/6/2010 Denaturation by pressure 18
SPC/E 1bar
The radial distribution function(simulation by MD)
*
333Argon
( y )
SPCE 15 kbar
2
(r) 2
)
2
(r)
Tetrahedal structure
1
g( g(r)
1
g(
111HexagonalStructure
01 2 3
00
5/6/2010Desnaturalización en frío
AFA ‐ Rosario 2009 19
1 2 3
r / σ
1 2 3
r / σ1 2 3
r / σ* Berendsen, Grigera. & Straatsma J. Phys Chem. 91, 6269‐6271. (1987)
G ‐ Decomposition4
Freundlich 1Gaussian 1 Tetrahedrical
3Hexagonal
Gaussian 2 Freundlich 2Gaussian 3
2
g Gaussian 5 Sigmoid Long distance
The sum = g(r)2 The sum = g(r)
1
0
5/6/2010 Denaturation by pressure 20
0 1 2 3 4 5 6r / σ
3
MD G-Decomposition
3
2
r)g(
r
1
1 2 3 40
5/6/2010 Denaturation by pressure 21
1 2 3 4
r / σ
2 5
3.0 g(r)
1 5
2.0
2.5
1.6 2 54
0 51.0
1.52.54
0.0
0.5
113
57 p / kbar
2 2
0 1 2 3 4
79
/
2.2
5/6/2010 Denaturation by pressure 22
r / σ
Order parameterpPr =1 Pure tetrahedralPr=‐1 Pure hexagonal. Pr= 1 Pure hexagonal
0.8
0.4
0.6
ussi
an
0.2
Ga
(Gaussian 1)A
5/6/2010 Denaturation by pressure 231 2
0.0
r / σ
0.2
0.1
0.2 water
argon
0.0
-0.1
Pr Crossover
0 3
-0.2P Crossover
-0.4
-0.3
0 2 4 6 8 10-0.5
5/6/2010 Denaturation by pressure 24
0 2 4 6 8 10
p / kbar
• The order parameter tell us the range in which water start to lose its characteristic properties.
• We will show our data on nonpolar solutes aqueous systems by Molecular Dynamics
• The question is: Can we reproduce the effects pressure and temperature on hydrophobic interaction?
5/6/2010 Denaturation by pressure 25
Lennard‐Jones particles in SPC/E water
0 8
1.0
e
0 6
0.8st
er S
ize
0.4
0.6
ed C
lus
DiameterConcentration
0.2
orm
aliz
e
0.0
No
2 3 4 5 6 7 8 9
Ratio Area LJ/SPCE*100
5/6/2010 Denaturation by pressure 26Ferrara, MacCarthy & Grigera J. Chem. Phys. 127, 104502 ‐1‐5 (2007).
Lennard‐Jones particles in SPC/E water0 80.8
Size
0.6
lust
er
0.4
Mea
n C
0 2lized
M
0.2
Nor
mal
200 250 300 350 400 450 5000.0
N
5/6/2010 Denaturation by pressure 27
T / KFerrara., MacCarthy & Grigera J. Chem. Phys. 127, 104502 ‐1‐5 (2007).
Lennard‐Jones particles in SPC/E water
50 Ferrara C.G., MacCarthy A. N. & Grigera J.R. J. Chem. Phys. 127, 104502 ‐1‐5
40
r Siz
e (2007).
30
Clu
ster
20
erag
e C
10Ave
0 1 2 3 4 50
5/6/2010 Denaturation by pressure 28
Pressure / kbar
1000Lennard‐Jones particles in SPC/E water
800
1000
0 4
0.6
0.8
an C
lust
er S
ize
800
ar 200 250 300 350 400 450 5000.0
0.2
0.4
Nor
mal
ized
Mea
600
P / b
a T / K
400P
200
300 400 5000
5/6/2010Cold denaturation
29
300 400 500T / KFerrara C.G. & Grigera J.R.
(Unpublished)
.
This simple system in which the onlyThis simple system, in which the only interaction present is the hydrophobic effect, shows a behaviour quite similar to what we can see in the proteins.can see in the proteins.
• Consequently, we consider that this q y,mechanism provided a coherent explanation f th d t ti b li dof the denaturation process by cooling and
pressure .
5/6/2010 Denaturation by pressure 30
We have made MolecularWe have made Molecular Dynamics simulation underDynamics simulation under pressure of different proteins.pressure of different proteins. We will show some results on apomyglobin
5/6/2010 Denaturation by pressure 31
RMSD = Root Mean Square of all α‐carbon atoms respect to the initial conformation
J
Apomyoglobin
5/6/2010Cold denaturation
32
Total Solvent Accessible SurfaceMD simulation of solvated apomyolgobin
1 b
MD simulation of solvated apomyolgobin(Chara , McCarthy , Ferrara , Caffarena & Grigera . Physica A 388, 2552, 2009).
‐‐‐‐‐ 1 bar‐‐‐‐‐ 3 kbar
3 kbar (extrapolated non‐linear regression)1 bar (average)1 bar (average)
5/6/2010Desnaturalización en frío
AFA ‐ Rosario 2009 33
Solvent Accesible Surface
MacCarthy & Grigera. B. B. Acta‐ Proteins and Proteomics 1764 506–515(2006)and Proteomics. 1764 506 515(2006)
5/6/2010 Denaturation by pressure 345/6/2010Cold denaturation
34
5/6/2010Cold denaturation
35
l iConclusion
The disturbance of the hydrogen bond y gnetwork by pressure or temperature alter the hydrophobic interaction (thealter the hydrophobic interaction, (the main driving force to maintain the native folding of proteins) and, therefore induce the denaturationtherefore, induce the denaturation.
5/6/2010 Denaturation by pressure 36
• MacCarthy A N & Grigera J R Effect of Pressure on the Conformation of
Recent related publication on the subject• MacCarthy A. N. & Grigera J.R. . Effect of Pressure on the Conformation of
Proteins. A Molecular Dynamics Simulation of Lysozyme J. of Mol. Graph. and Mod.. 24, 254–261 (2005)M C h A N & G i J R P D i f A l bi A• MacCarthy A. N. & Grigera J.R. Pressure Denaturation of Aapomyoglobin. A Molecular Dynamics Simulation Study. Bioch. Bioph. Acta‐ Proteins and Proteomics. 1764, 506–515(2006)
• Ferrara C.G., MacCarthy A. N. & Grigera J.R. Clustering of Lennard‐Jones particles in water: Temperature and pressure effects. J. Chem. Phys. 127, 104502 ‐1‐5 (2007)
• Chara O., MacCarthy A.N., Grigera J.R. Water behavior in the neighborhood of hydrophilic and hydrophobic membranes: Lessons from molecular dynamics simulation. J. Biol. Phys.s 33, 523‐539 (2007)
• Chara O., McCarthy A.N., Ferrara C.G., Caffarena E.R. & Grigera J.R. Water behavior in the neighborhood of hydrophilic and hydrophobic membranes: Lessons from molecular dynamics simulation Physica A 388, 2552‐2449 (2009). f y y , ( )
• Grigera J.R. & McCarthy A.N. The behavior of the hydrophobic effect under pressure and protein Denaturation. Biophys. J. 98; 8, : 1527; DOI: 10.1016/j.bpj.2009.12.4298 (in the press).
5/6/2010Cold denaturation
37
10.1016/j.bpj.2009.12.4298 (in the press).• Chara O., McCarthy A.N. & Grigera J.R. Crossover between tetrahedral and
hexagonal structures in liquid water (submitted).
ColaboradoresCollaboratorsIn the development of this work have participated:
Dr Osvaldo Chara
work have participated:
Dr. Osvaldo Chara
Dr Andrés N McCarthyDr. Andrés N. McCarthy
Dr Ernesto R CaffarenaDr. Ernesto R. Caffarena
Lic Carlos G FerraraLic. Carlos G. Ferrara
5/6/2010Desnaturalización en frío
AFA ‐ Rosario 2009 38IFLYSIB, La Plata, Argentina
The work was supported (but not
l ) btoo generously) by
‐The University of La.‐The University of La Plata
The National Research Council of
i (CO C )Argentina (CONICET)
‐ National Agency for‐ National Agency for Promotion of Science and Technology of Argentina (ANPCyT)
5/6/2010 Denaturation by pressure 39La Plata, Cathedral
5/6/2010Cold denaturation
40