Protein Folding Energetics, Kinetics and Models Oznur Tastan [email protected] Graduate Student...

Protein FoldingEnergetics, Kinetics and Models

Oznur Tastan [email protected]

Graduate Student

Carnegie Mellon University

03/23/2006 Molecular Biophysics III: Spring 2006: Oznur Tastan 2

Lecture Outline

• Introduction: What is protein folding and why it is a problem?

• Globular Protein Folding Models

• Detection and characterization of denatured states, intermediates, such as the molten globule and comparison to folded states

• Kinetics and pathways

• Membrane Protein Folding Models

• 2-stage and 3-stage hypothesis

• New long range interaction hypothesis

• Summary


Lecture Outline








• Summary


Our focus

in this lecture

http://www-nmr.cabm.rutgers.edu/academics/biochem694/2006BioChem412/Biochem.412_2-24-2006lecture.pdf


Folding is encoded in the amino acid sequence. Native state is the minimum energy state.

Addition of mercaptoethanol and urea

Removal of mercaptoethanol and urea

Native, catalytically active

state.

Native,catalytically active

state. Refolded correctly!

Unfolded; catalytically inactive.

Reduced disulfide bonds.

1/105 random chance

Anfinsen, 1973.

Anfinsen’s Experiment


The Protein Folding Problem:Writing the book of Protein Origami

http://www.idi.ntnu.no/grupper/KS-grp/microarray/slides/drablos/Fold_recognition/sld006.htm

Now collapse down hydrophobic core, and fold over Helix A to the

dotted line. Bring charged residues of ‘A’ into close proximity

of ‘B’ ..?

+membrane


How does a protein fold?Levinthal’s Paradox

Entropy

En

erg

y

Protein folding cannot be random-walk.

Simplest case: random-walk• Assume a chain of 100 amino acids.• Allow only 3 conformations.

- Possible conformations = 3100 ~ 1048

• Assume bond rotation rate 1014 sec.

- Reaching the native state would take: 1026 years ! Longer than the age of the universe!

Dill & Chan, 1997Levinthal, 1968


Why is protein folding problem difficult?

• Folding can be very fast, millisecond to second (slow folding is easier)

• Small energy changes between the denatured state to the native state ( 1-15 kcal/mol) - equivalent to the strength of a few hydrogen bonds

• The states populated along pathway are ensembles of structures

Comparison from multiple complementary techniques are required.


The Three Protein Folding Models

Framework model

Nucleation condensation

model

Hydrophobic collapsemodel

http://www.makro.ch.tum.de/users/BFHZ/Scheibel/Scheibel%202003%20Bordeaux-1.pdf


Lecture Outline








• Summary


The Native State

• A complex balance between: 1) Short-range local interactions

-intrinsic conformational preferences of the amino acids

2) Medium-range interactions

-stabilizing regions of secondary structure 3) Long-range interactions

- tertiary interactions determining the global fold

• Generally single conformation (with small fluctuations around the mean torsion angles).


Random Coil and Denatured State

“Φ,Ψ angles of each residue is sterically independent”There should not exist any

non-local interactions.

Flory’s isolated pair hypothesis

Rg= RgNv

N = Length (Residues) v = Solvent viscosity

parameter

Rg values of 28 denatured proteins

obeys the Flory’s power law.Flory, 1969.

Rg values determined by SAXS

Sosnick, T.R., et al. 2004


Testing the random coil statistics

Fitzkee, N.C. and Rose, G.D. 2004

Despite 92% of the native structure kept, random coil statistics are obtained.

Simulated Rg follows the power law.

33 proteins

Number of residues

For a protein ≈8% of the residues are varied; the remaining ≈92% of the residues remained fixed in their native conformation.


The Denatured StateDoes Flory’s hypothesis hold?

Conformations of polyalanine chains are enumerated to test the hypothesis.

Pappu et.al 2003.

+ ={A,G,M,R,L,F,E,K,Q}

* = {J,P,O,I,o}

Flory’s hypothesis is not valid for polypeptide chains. Backbone conformations are limited by

additional steric clashes.


Can we get a structure of the “denatured state”?

When the folded state breaks down:

1. The dispersion of all resonances decreases: - Extensive overlap of peaks.

2. Greater dynamic motions between residues: - Weak or eliminated NOEs between protons.

3. Ensemble of conformations: - Each NMR parameter reflects an average over a dynamic

ensemble of conformations.

Attainment of a high-resolution structure is not possible in the non-native state.


NMR as a tool to study denatured states

67891011121H Chemical Shift (ppm )

105

110

115

120

125

130

15 N

Ch

emic

al S

hif

t (p

pm

)

7.08.09.010.0

112

114

116

118

120

122

124

126

128

1H Chemical Shift (ppm )

15N

Ch

emic

al S

hif

t (p

pm

)

Folded lysozyme: Unfolded lysozyme:

For small proteins, all backbone resonances can be resolved even in the denatured state.


1. Measurement of NMR parameters in 15N-labeled unfolded protein

2. Comparison of NMR parameters

- unfolded with random coil parameters (sources:

- statistical analysis from unfolded peptides - random coil models (e.g. polymer model, Model-free analysis, Flory etc.)

- unfolded with folded state parameters- different degrees of unfolded states

•Chemical shifts

•Relaxation rates

•Heteronuclear NOE

•Dipolar couplings

•Scalar couplings

Which and how do we use NMR parameters?







•Chemical shifts

•Relaxation rates



•Scalar couplings



Persistence of native-like topology in the denatured states

Denatured proteins can preserve long range ordering, in conflict with the random-coil models.

Unf

olde

d

Folded

Shortie. et. al. 2001

SNase N-H Dipolar couplings







•Chemical shifts

•Relaxation rates



•Scalar couplings



Residue

0 25 50 75 100 125

R2

[s-1

]

0

2

4

6

8

10

12

Residue

0 25 50 75 100 125

Urea Water

Residue

0 25 50 75 100 125

R2

[s-1

]

0

2

4

6

8

10

12

Residue

0 25 50 75 100 125

Urea Water

1

||

int)(N

j

ji

rinsic eRiR

Random Coil Model of Segmental Motion

Residue

0 25 50 75 100 125

R2

[s-1

]

0

2

4

6

8

10

12

Residue

0 25 50 75 100 125

Urea Water

0

20 ||

x

b

xi

Ae

+ Gaussian Distributions of Deviations

There are six clusters of residual structure in WL-SME.

1.

2.

3.

4.5.

6.

Residual structure in lysozyme

WL-SME in urea WL-SME in water

Klein-Seetharaman, 2002.


A single point mutation, W62G in cluster 3, disrupts all clusters in reduced and methylated lysozyme.

Experiment: Mutation of W62

Klein-Seetharaman, 2002.


The Molten Globule(MG) State

Molten globule is characterized by

1. Absence of specific tertiary contacts

2. presence of some secondary structure

3. Native-like compactness

4. Presence of hydrophobic core

Example: -lactalbumin

Molten globule observed in low pH




2. Presence of some secondary structure


4. Presence of hydrophobic core Kuwajima, K. 1989.

native pH5.4

MG-state pH2

unfolded state (in 9M urea,pH2)




2. Presence of some secondary structure


4. Presence of loosely packed hydrophobic core.

Rg

Native 15.7+0.2

MG 17.2+0.3

Unfolded 30.0+0.7

Katoka, 1997.


The Molten GlobuleSignificance for Protein Folding Mechanism

Disordered polypeptide collapse into the molten globule. According to one view,

http://www.bmb.psu.edu/courses/bmb401H/Chapter7and8.pdf


How do small single-domain proteins fold?

• 20 small proteins(< 100 aa) are showed to fold :

– simple two-state folding kinetics

– show variation in their folding rates(microseconds to seconds)

– all structures has to pass the transition state in order to reach the native state

Dobson, 2003


Kinetics of Two-State Folding

ΔGŦ-D = GŦ - G°D = - RTlnkfold ΔGN-D = G°N – GŦ = - RTlnkunfold

λ = kfold + kunfold

[GdnHCl]

Chevron Plot of CI2

ln λ

kfold kunfold

An indicator of 2-state kinetics.


Ф-value AnalysisCharacterization of the Transition State(TS)

Ф=1: site of mutation is native-like in TS.

Ф=0: site of mutation is unfolded in TS.

Fractional Ф value: partial structure in TS.

TS cannot be isolated or studied directly.

Reproduced form

Systematically introduce mutations in the native protein.

Infer structure of TS from the energetics of the folded state (mutant versus wild-type).

Ф = ΔΔGŦ-D / ΔΔGN-D


Transition State Analysis:Case Study I: Chymotrypsin inhibitor(CI2)

In the transition state of CI2 three residues with Ф-values >0.5 come

together :A16,L49,I57.

A hydrophobic core supporting the nucleation-condensation

mechanism


Complex pathwaysCase study II: hen lysozyme

Thus cannot be approximated with simple 2-state kinetics.

Most proteins (>100 aa) fold with observable intermediates.

lysozyme

Dill & Chan et al.1997


Understanding how lysozyme folds

Radford,et.al,1992

α

α

β

β

β

α

α

α

Alpha and beta domains are two distinct folding units.

α

HX + NMR

α

β


The details?

α domain is structured independently of the β

domain in the early stages of folding.

HX labeling &

EMS

Far-UV CD

Large secondary structure is formed

within the milliseconds of folding.

unprotected

β

α

Dobson,et.al. 1994

Single exponential, the α domain forms before the near UV develops.

The tertiary contacts are not fixed yet.

Near-UV CD


Intrinsic Trp flouresence

A change in the some or all of the Trp occurs in early collapse in later

intermediates and on formation of the native structure.

Quenching of flouresence by iodine

Exclusion of water in the early stages of folding

Binding of ANS

Maximal emission in the early stages.

A relatively loosely packed, condensed state exists in the

early stages of reaction.Dobson,et.al. 1994


Folding pathway of lysozyme

Very rapidly alpha domain forms.

Hydrophobic interior develops. Few tertiary interactions.

Protective structure evolves. Dynamic and

fluctuating beta domain.

Alpha and beta domains are stabilized

Major >30%Minor ~10%

Dobson,et.al. 1994


Complementary approaches are essential! Dobson, 1998.


Summary

No clear unifying view of protein folding has yet emerged.


Membrane Protein FoldingModel systems

α-helical bundles β-barrels

The lipid environment

OmpABacteriorhodopsinMammalian Rhodopsin


Denaturation of bacteriorhodopsin

Effects of Urea and Guanidinium Hydrochloride:

almost none, not even on tertiary structure.


Denaturation of Bacteriorhodopsin

NativeSDS

Formic acid

Secondary structure remains even in SDS.


Why is it so difficult to disrupt secondary structure in membrane

proteins?


Thermodynamic considerations

The engaging of polar backbone in H-bonds is favorable.

White & Wimley,1999.


Refolding of Bacteriorhodopsin in the lipid bilayers

BR can assemble in to the native structure when helices are inserted into the membrane independently.

Retinal reconstituted:

%90 of the activity regenerated.

Refolded: fragments have near-identical

helix content.

1. C-1 and C-2 in SDS 2. C-1 and C-2 +lipid 3. Retinal omitted.

Popot, 1987.

C-1C-2


The two-stage hypothesis: Independent helix intermediate

1st Stage:

Helix folds Independently

2nd Stage:

Final packing and interactions between

the helices are formed.New 3rd Stage:

Biding of prosthetic groups, folding of loops,

oligomerization…

Two stage hypothesis may not hold in all cases.

Engelman &Popot, 1990.Engelman & Popot, 2003. Figures from Klein-Seetharaman,2005.


HR unfolded with AFM

Cooperative unfolding barriers are observed, in conflict with the 2-stage hypothesis.

Helix G unfolded in two stepsHelixF

Helix E unfolds with helix D

Short cytoplasmic segment

Helix C

B-C loop

Helix B

Helix A unfolds in two steps

Part of

helix E

Cisneros, 2005.


Folding Core Prediction of Rhodopsin

Agreement with the mutational data (>%90 ).

Evidence for the significance of long-range interactions in the folding of

rhodopsin.

Gaussian Network Model (GNM) FIRST

Folding core lies in the EC-TM domain interface.

Rader. et.al 2004


New Model: Long Range Interaction intermediate

Klein-Seetharaman,2005.


References• Anfinsen, C.B. (1973) "Principles that govern the folding of protein chains." Science 181 223-230.

• Cisneros, D.A., D. Oesterhelt, and D.J. Muller, Probing origins of molecular interactions stabilizing the membrane proteins halorhodopsin and bacteriorhodopsin. Structure, 2005. 13(2): p. 235-42.

• Dobson, C.M.Sali A., and Karplus, M., "Protein Folding: A Perspective from Theory and Experiment", Angew. Chem. Int. Ed. Eng. 37, 868-893 ( 1998).

• Dobson, C.M. "Protein Folding and Misfolding", Nature 426, 884-890 ( 2003).• Dobson, C.M., P.A. Evans, and S.E. Radford, Understanding how proteins fold: the lysozyme story so far. Trends Biochem Sci,

1994. 19(1): p. 31-7. • Engelman, D. M., Chen, Y., Chin, C. N., Curran, R., Dixon, A. M., Dupuy, A, Lee, A., Lehnert, U., Matthews, E., Reshetnyak, Y., Senes, A.,

Popot, J-L. “Membrane Protein Folding: Beyond the Two Stage Model” FEBS Lett. (2003) 555:122-5.

• Flory, P. J. (1969) Statistical Mechanics of Chain Molecules (Wiley, New York).• Fitzkee, N.C. and Rose, G.D. (2004). Reassessing random-coil statistics in unfolded proteins. Proc. Natl. Acad. Sci. 101: 12497–12502.• Klein-Seetharaman, J., Oikawa, M.,Wirmer, J., Duchardt, E., Ueda, T., Imoto, T., Smith, L.J., Dobson, C. and Schwalbe, H. (2002) Long-Range

Interactions within a Non-Native Protein. Science 295, 1719-1722. • Klein-Seetharaman, J. (2005) Dual role of interactions between membranous and soluble portions of helical membrane receptors for folding and

signaling. Trends in Pharmacological Science 26(4), 183-189 • Kuwajima, K. (1989). The molten globule state as a clue for understanding the folding and cooperativity of globular-protein structure. Proteins:

Struct. Funct. Genet. 6: 87–103.• Kataoka, M., Y. Hagihara, K. Mihara, and Y. Goto. (1993). Molten globule of cytochrome c studied by the small angle X-ray scattering. J. Mol.

Biol. 229:591-596.• Radford, S. E., Dobson, C. M. & Evans, P. A. The folding of hen lysozyme involves partially structured• intermediates and multiple pathways. Nature 358, 302-307 (1992).• Pappu, R. V. , Srinivasan, R. & Rose, G. D. (2000) Proc. Natl. Acad. Sci. USA 97, 12565-12570.• Popot, J-L and Engelman D.M."Membrane Protein Folding and Oligomerization: The Two-Stage Model“ Biochemistry (1990), 29 (17), 4031-7.• Popot J.L., Gerchman S.E., Engelman D.M. (1987) Refolding of bacteriorhodopsin in lipid bilayers. A thermodynamically

controlled two-stage process. J. Mol. Biol. 198:655-76• Shortle D, Ackerman MS. (2001) Persistence of native-like topology in a denatured protein in 8 M urea. Science. Jul 20;293(5529):487-9. • White S. H. and Wimley, W. C. (1999). Membrane protein folding and stability: Physical principles. Annu. Rev. Biophys. Biomol. Struct. 28:319-

365. • http://www.otago.ac.nz/humannutrition/dietetics/gfx/philosophy.jpg, March 22, 2006.

• http://www-nmr.cabm.rutgers.edu/academics/biochem694/2006BioChem412/Biochem.412_2-24-2006lecture.pdf, March 22, 2006.

• http://www.makro.ch.tum.de/users/BFHZ/Scheibel/Scheibel%202003%20Bordeaux-1.pdf, March 22, 2006.


Acknowledgements

Dr. Judith Klein-Seetharaman

Dr. Sanford Leuba

&

The class of MB3 (Spring 2006)


QUESTIONS?

http://www.otago.ac.nz/humannutrition/dietetics/gfx/philosophy.jpg

Protein Folding Energetics, Kinetics and Models Oznur Tastan [email protected] Graduate Student...

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