1

Protein Folding

What stabilizes a given fold?

U F

Thermodynamic Forces that Stabilize theFolded State

Ionic, H-Bonds, Van der Waal’s Forces, Hydrophobic Effect

U F

2

Thermodynamic interactions that stabilizethe folded state of a protein

Ionic bonding (salt bridges)

Thermodynamic interactions that stabilizethe folded state of a protein

Hydrogen bonds in a protein molecule

hydrogen bondhydrogen bond

3

Thermodynamic interactions that stabilizethe folded state of a protein

van der Waal’s forces

Leonard-Jones Potential

Thermodynamic interactions thatstabilize the folded state of a protein

The hydrophobic effect

4

How much more stable is the foldedstate than the unfolded state?

U F

NOT MUCH!

How do we understand this equilibrium?

Keq =

Recall 3 Cases:Keq > 1 rxn favors folded stateKeq < 1 rxn favors unfolded stateKeq = 1 equal mixture of U and F

U F

ΔG° = -RTlnKeq

G = Gibbs Free EnergyΔ = Change

3 Cases:ΔG ° < 0 rxn favors folded stateΔG ° > 0 rxn favors unfolded stateΔG ° = 0 equal mixture of U and F

5

Components of ΔGΔG = ΔH - TΔS

H Enthalpy: a measure of heat ΔH < 0 gives off heat; exothermic ΔH > 0 absorbs heat; endothermic ΔH = ∑BEbroken - ∑BEformed

S Entropy is a measure of disorder ΔS > 0 system is more disordered

ΔS < 0 system is more ordered ΔS = Sfinal state - Sinitial state

2nd Law of ThermodynamicsΔSTotal > 0

ΔStotal = ΔSsurroundings + ΔSsystem

The total entropy of the universe is always increasing.

6

Some chemical reactions “go”because ΔH is negative (exothermic)

G

reaction coordinate

Reactants

HEATHEAT

Products

But . . . not all reactions that go spontaneously areexothermic (give off heat).

H20 (liquid)H20 (solid)Above 0° C

ΔG < 0

Ice melting is an endothermic process

ΔG = ΔH - TΔSΔH > 0 ΔS > 0

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H20 (solid)H20 (liquid)

ΔH < 0 ΔS < 0

Below 0° C

ΔG < 0

ΔG = ΔH - TΔS

Heat is given off when bonds are formed(i.e. water freezing is exothermic)

No: the heat released upon freezingcreates more disorder in the surroundings

Does this violate the 2nd law of thermodynamics?

ΔH < 0 ΔSsystem < 0, but ΔStotal > 0

8

OH

H

O

H

H

O

H H

OH

H

O

H

H O

HH

OH

HO

H

H

O

HH

O

H

H

O

H

H

Protein Folding

U F

ΔSOH

H

O

HH

O

H

H

O

H

H

OH

H

O

H

H

O

H

H

O

H

H

Water plays a large role in protein folding.

orderedwater disorderedwater

disorderedpolypeptide orderedpolypeptide

?

The Hydrophobic Effect

OH

H

O

H

H

O

H H

OH

H

O

H

H O

HH

OH

HO

H

H

O

HH

O

H

H

O

H

H

CH2

H2C

CH2

H2C

CH2

H2C

H3C

CH3O

H

HO

H

HOHH

OH

H

O

H

HO

H

H

O H

H

O

H

H

O

HHO

HH

O

H

H

H2C

CH2

H2C

CH2

H2C

CH2

CH3

H3C

OH

H

OH H

O

H

H

OH

H

O

H

H

O

H HO

H

CH2H2C

CH2H2C

CH2H2C

H3C

CH3O

H

H

OHH

O

H

H

O H

H

O

H

H

O

HHO

H

H2C

CH2

H2C

CH2

H2C

CH2

CH3

H3C

H

H

OH

H

O

HH

O

H

H

O

H

H

OH

H

O

H

H

O

H

H

O

H

H

disorderedwater

+

orderedwater

The release of disordered water upon aggregation causes anincrease in the entropy of the universe (ΔST > 0).

9

Kinetics - what path does the unfoldedchain take to get to the folded state?

Levinthal’s Paradox:

- each amino acid has 9 conformations(Ramachandran plot)

- Conversion between conformations:10-12sec

- 150 aa chain

~ 10150 conformationsTo search all 10150 conformationsrequires 10138 sec ~ 10130 years!!

But - protein folds in ~0.1-~1000 sec. HOW?

Speculation: Folding process is directedto avoid irrelevant/incorrect conformations

One idea : Domains guide folding

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Denaturation is often irreversible.

Humpty Dumpty’s Fate:

Concentration oftotal protein insidecells = 200-300g/LCellular interiorsare 20-30% volumeoccupied bymacromolecules(Red blood cellscontain 350g/L ofhemoglobin alone)

Proteins in cells are densely packed.

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Chaperones provide a secluded environment forproteins to fold in.

Anfinsen’s Cage

Take Home Messages

• The side chains of the amino acids determine theshape and properties -- and thus the functions ofproteins.

• Folded proteins are marginally stable. Thehydrophobic effect plays a huge role in the stability ofthe folded protein.

• YOU MUST UNDERSTAND how to apply theequation: ΔG = ΔH - TΔS

• Proteins do not sample every possible conformationin the time it takes them to fold, and sometimesfolding must be catalyzed.