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:

Under that conditions the PT diagram is elliptic.

pPressure denaturation

Colddenaturation

T

denaturationNative


8

Heat denaturation

4000p/bar

3000

2000 Chymotrypsinogen (pH 2.07)

1000

0 10 20 30 40 500


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


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


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



, y , ( )

Hydrophobic Interactionyd op ob c te act o

ΔSIh>0

ΔSHh<0

Approx 1kcal/mol (4 kJ/mol)



.

• 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?


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


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


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 ?


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



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


0 1 2 3 4 5 6r / σ

3

MD G-Decomposition

3

2

r)g(

r

1

1 2 3 40


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


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


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?


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


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


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


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 .


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


RMSD = Root Mean Square of all α‐carbon atoms respect to the initial conformation

J

Apomyoglobin


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)



Solvent Accesible Surface

MacCarthy & Grigera. B. B. Acta‐ Proteins and Proteomics 1764 506–515(2006)and Proteomics. 1764 506 515(2006)


34


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.


• 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).


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


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


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J. RúlR aúl GiGrigera · Free energy of denaturation ΔG P 0 Cold denaturation Heat denaturation...

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