Biophysik der Moleküle

4. Vorlesung Rädler WS 2010

28. Oct. 2010

Protein folding- Afinsen hypothesis- hydrophobic interaction

Gaub/SS 2005 BPM §1.3 2

Protein Unfolding: Sushi Restaurant

When foods with proteins are exposed to heat and certain chemicals (such as vinegar), they turn white.

1. Distinguish salmon roe from imitation salmon

roe by dropping into hot tea.

2. Mackerel is pickled in vinegar for

preservation.

Gaub/SS 2005 BPM §1.3 3

Nobel Prize for Chemistry in 1972

C. Afinsen 1916-1995

The Thermodynamic Hypothesis (Afinsen 1973)

„the native state is thermodynamically stable“

=> the sequence alone

determines 3D structure!

ribonuclease A

loop(usually exposed on surface)

alpha-helix beta-sheet

Afinsen‘smodel protein:ribonuclease A

Ribonuclease kann durch Oxidation (Spaltung der S-S Bindung)denaturiert werden

o) Nofür t iche

Ribonuclea se

Abb. 3.12: Die zwei Zuslände der Ribonuklease:

links: KomDakt! Funktionsfom

rechts: Oenatudert! Form

b )0eno tu r ie r te

Ribonu cleose95

HH

65

Das Enzym hat 8 s-s Bindungen. Im Prinzip könnten 56 verschiedene Zustände (Isomere) gebildet werden. Es gibt aber offenbar nur einen Zustand niedrigster Energie.

7

Folding of RNAse A in the test tube

denaturation renaturation

Incubate proteinin guanidine

hydrochloride(GuHCl)or urea

100-folddilution of proteininto physiological

buffer

Anfinsen, CB (1973) Principles that govern the folding of protein chains.

Science 181, 223-230.

- the amino acid sequence of a polypeptide is sufficient to specify its three-dimensional conformation

Thus: “protein folding is a spontaneous process that does not require the assistance of extraneous factors”

(aggregation)

Folding of proteins in vivo is promoted by chaperones

this bears only on the rate of folding

However:

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What drives protein folding?

Minimization of G=E-TS+PV

Minimize the solvation energy.Decrease the conformational entropy.

10

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GFP Fluoreszenz Siehe Biophysik F-Praktikum

Other techniques to probe unfolding

High-resolution techniques (local):

• FTIR

• Fluorescence

• NMR

• UV absorption

Low-resolution techniques:

• SAXS

• DLS

Which forces are dominant in protein folding ?Local vs. non-local interactions

Nonlocal interactions drive collapse transition, whereas local interactions drive helix transitions.

16

Early model in which protein folding was proposed to be

driven by ion-paired hydrogen bonding among side chains

(Mirsky& Pauling, 1936; Eyring & Stearn, 1939)

disproven by Jacobsen and Linderstrom-Lang

Electrostatic Contributions

• !i=(zie/4"#o)(1/r2) coulomb potential

• Sensitive to pH and ion concentrations

• pH determines total charge (pI)

• Ionic strength determines effective range of interactions

• Ion pairs contribute 1-3 kcal/mol (on surface)

• Ion pairs generally destabilizing if buried (cost up to 19 kcal/mol/ion to completely bury

• Ion pairs contribute ~5-15 kcal/mol per 150 aa’s

The Kauzmann Hypothesis

„hydrophobic interactions determine the thermal stability of the native state“

* non-polar solvents denature proteins* „unusual“ temperature dependence: (stability decreases at high AND low temperatures)* protein stability follows same salt dependence as lyotropic (Hofmeister) series

Key arguments

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Determination of protein stability.

• This can be measured with a variety of tools including, microcalorimetry, spectroscopy, and enzyme function.

• The transition can be accomplished with heat or denaturants.

• The area under the curve gives $H which agrees with measurements based on the van't Hoff equation

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Denaturants• Temperature

• pH– Change the ionization state of critical residues

• Detergents– Bind strongly to the unfolded protein

• High concentrations of water soluble organic substances– Aliphatic alcohols. These disrupt the water structure

• Ionic or polar denaturants including urea and guanidinium

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Denaturants: The Hofmeister Series

• The ability of an ion to stabilize a protein follows the Hofmeister series

• Anions! SO4

2->H2PO4->CH3COO->Cl->Br->I'->ClO4

->SCN-

• Cations

NH4+,Cs+,K+,Na+>Li+>Mg2+>Ca2+>Ba2+

! ! ! >guanidinium>urea

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Thermal stability of RNase A as a

function of salt

• This illustrates the effect on protein stability for many commonly used salts.

• Potassium phosphate and ammonium sulfate stabilize proteins which accounts for their frequent use in protein purification.

From Voet and Voet second edition

The hydrophobic effect

water forms cluster with coordination number 4

proteins are surrounded bya shell of structured water

!

µK ,W

= µK ,W

0 + RT ln xK ,W

Solubility and partition function

!

µK

0"µ

W

0 = HK

0"H

W

0( ) "T SK

0" S

W

0( )!

µW

= µK

!

ln xW

* =µK

0"µ

W

0( )RT

chemical potential,!, and partition coefficient,x of oil molecule in water (w) and oil (K)

at equilibrium:

!

"µ = µW

0#µ

K

0 = 2.44 + 0.88nC

enthalpic entropic

[kcal/Mol]

The entropic change (cost of inducing water order) dominates over the enthalpy change (gain in intermolecular interaction), which is also negative.

!

"µ = #4.2 + 0.825nC

for alcoholes

for alcanes

!"#$%&'()*%+,-"#./*0#).1&).2&"3#)4*,,#$,0&5#).+).6*,,#$.7#+.89:.;

StoffµW

0

! µK

0

in kcal/Mol

HW

0! H

K

0

in kcal/Mol

SW

0! S

K

0

in cal/Mol K

C2H

63.9 -2.5 -21

C3H

84.9 -1.7 -22

C4H

105.9 -0.8 -23

HW

0

! HK

0!"#$%&'$()*$+,-$.&%$/%&$.%-$0/%-+,1-2'3$%&'%"$(45"$.%"$67$84'$9$'):1$;$+-%&;%-.%'.%$7<-=%>$?)$.&%"%-$7%-#$'%3)@8$&"#A$&"#$.&%$0/%-+,1-2'3$%&'$%B4#1%-=%-$C-4D%*>$?&%$E)'F.%-F7))5"FG'D&%12'3$D;&":1%'$.%=$67F(45%9,5$2'.$7)""%-$&"#$/%#-<:1#5&:1>$

$!"#$'%3)@8A$.)"$/%.%2#%#A$%"$H'.%#$%&'%$G/')1=%$.%-$I)-@%55%'$J'#-4I&%$/%&$.%-

0/%-+,1-2'3$.%"$67$84'$9$'):1$;$"#)K>$LJ&"/%-3"#-29#2-$.%"$7)""%-"MSW

0

! SK

0

!µ = µW

0" µ

K

0= 2.44 + 0.88n

C%=I&-&":1N ':NO)15$.%-$6415%'"#4P)#4=%

Hydrophobic Effect• At normal temp’s the “hydrophobic effect” is entropic

water molecules form ordered structures around nonpolar

compounds

• Hydrophobic residues collapse in to exclude water

• Additional forces can then stabilize (vdw, h-bond,intrinsic properties)

• Hydrophobic effect is dependent on temperature (unstable at high AND low temp).

Thermodynamic considerations• Protein stability is composed of two components.

% % $G = $H-T$S

• There is a complex temperature dependence for $H and T$S which means that the contribution of the enthalpic and entropic terms changes with temperature.

• This temperature dependence arises from the anomalously high change in heat capacity on transferring hydrophobic compounds into water. This is the hall-mark of the hydrophobic effect and arises from the water-ordering.

Heat Capacity

• The heat capacity influences both the temperature dependence of the enthalpy and entropy

• It is proportional to the buried non-polar surface area as are all of the thermodynamic parameters.

• The large heat capacity is indicative of a well ordered water structure around non-polar molecules in water as is evident from their partial specific volumes when dissolved in water

!

Cp ="H

"T=T"S

"T

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Temperature dependence of $G

• Thermodynamics of transfer of a hydrocarbon from liquid to aqueous solution.

• The temperature dependence is the result of different heat capacities of the two phases.

• The large changes in $H and T$S compensate so that $G is fairly constant with temperature

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Temperature dependence of $H and T$S continued

$H becomes more favorable at lower temperatures, whereas the entropic term becomes less favorable. This is consistent with an increase in the order in the water surrounding the non-polar molecule.

The water-ordering increases the interaction between solvent and solute and thus "enhances" the solubility that would occur in its absence. Even so, the interactions between solute and water eliminate hydrogen bonds within the water that cannot be compensated for by the ordering of the water.

Significantly the van der Waals interactions are greater in the pure water and solute than in the dissolved solute.

It is the loss of hydrogen bonds and van der Waals interactions that is the cause of the hydrophobic interaction.

$H is ~0 at

room temperature

Terms counterbalance

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Buried hydrophobic surface area• The buried hydrophobic surface area for a protein

correlates with the protein stability.

• Although it is difficult to predict the overall stability of a protein, it is possible to predict the worst case scenario that a mutation might produce based on changes in buried surface area.

• Occlusion of 1Å2 of hydrophobic surface area provides ~25 cal mol-1 of stability.

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$Cp vs $Anp for proteins

There is a linear correlation between the heat capacity change for protein unfolding and the buried non-polar surface area.

This relationship is identical to that seen for the transfer of hydrocarbons from aqueous solution to the pure liquid phase

From Livingstone JR, Spolar RS, Record MT Jr. Biochemistry. 1991 Apr 30;30(17):4237-44

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Protein Unfolding: Pressure?• 1895 Royer discovered that high hydrostatic pressure kills

bacteria.

• 1899 Hite uses pressure for milk preservation.

• 1914 Bridgman notices that egg white looks ‘cooked’ after pressure treatment.

Though it isn’t intuitive, proteins also unfold with pressure.

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High-Pressure SAXS Study

SAXS: shine X-ray on sample, look at scattering intensity vs. scattering angle.

• Guinier approximation: I~Io exp(-Rg

2/3)

Detect global size changes.

-> for pressure studies, this may give the most relevant information.

Minimization of Volume

More efficient packing is accomplished when small water molecules penetrate

the hydrophobic core. (10 basket balls and 1000 golf balls – pack the basket

balls clustered or separated. Which takes up less space?)

atmospheric P hydrophobic packing? unfolding?

Faltungsproblem

Konformation eines Proteins als Random Walk:

Gitter-Modell: Kleines Protein mit 100 Aminosäuren

=> Mögliche Konformationen: 3100" 1030

Interne Dynamik typ ns

&Zeit, um alle möglichen

Kombinationen durchzuspielen " 1021 s

Vergleiche: Alter des Universums " 1020 s !

Mother nature has no folding problem,but we do!

How many conformations are there in the Native state?

the number of sequences that have N native states decreases strongly

the HP model

molten globuleshave many configurations

The reason that only one native structure is encoded in the aminoacid sequence may be largely attributable to the hydrophobicinteraction; there are only a small number of ways to configurea chain to maximize the number of nonpolar contacts.

These forces are of a nature such that proteins should be tolerant of amino acid substitution, a given native structure should be encodable in many different sequences, and a large fraction of all possible sequences should fold to compact structured native states.

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Levinthal paradox

folding

denaturedprotein:

random coil1030 possibleconformations

Native protein1 stable

conformation

in vitro in vivo

folding

t = secondst = seconds or much less

Up To Date No Unified Folding Theory