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Folding. Judith Klein-Seetharaman Department of Structural Biology jks33@pitt.edu. Objectives of this Lecture. Overview Folding/Misfolding Anfinsen Levinthal Paradox Folding Models The denatured state The molten globule Two-state folding Deciphering complex folding pathways.

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Folding l.jpg

Folding

Judith Klein-SeetharamanDepartment of Structural Biology

jks33@pitt.edu


Objectives of this lecture l.jpg
Objectives of this Lecture

  • Overview Folding/Misfolding

  • Anfinsen

  • Levinthal Paradox

  • Folding Models

  • The denatured state

  • The molten globule

  • Two-state folding

  • Deciphering complex folding pathways

Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


Objectives of this lecture3 l.jpg
Objectives of this Lecture

  • Overview Folding/Misfolding

  • Anfinsen

  • Levinthal Paradox

  • Folding Models

  • The denatured state

  • The molten globule

  • Two-state folding

  • Deciphering complex folding pathways

Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


Overview l.jpg
Overview

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

Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


Objectives of this lecture5 l.jpg
Objectives of this Lecture

  • Overview Folding/Misfolding

  • Anfinsen

  • Levinthal Paradox

  • Folding Models

  • The denatured state

  • The molten globule

  • Two-state folding

  • Deciphering complex folding pathways

Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


Anfinsen s experiment l.jpg

Oznur’s slide:

Anfinsen’s Experiment

Addition of

mercaptoethanol and urea

Removal of

mercaptoethanol and urea

Native,

catalytically active state.

Refolded correctly!

Native,

catalytically active ribonuclease A

Unfolded;

catalytically inactive.

Reduced disulfide bonds.

1/105 random chance

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

Anfinsen, 1973.

Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


Objectives of this lecture7 l.jpg
Objectives of this Lecture

  • Overview Folding/Misfolding

  • Anfinsen

  • Levinthal Paradox

  • Folding Models

  • The denatured state

  • The molten globule

  • Two-state folding

  • Deciphering complex folding pathways

Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


How does a protein fold levinthal s paradox l.jpg

Oznur’s slide:

How does a protein fold?Levinthal’s Paradox

  • 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!

Simplest case: random-walk

Energy

Entropy

Protein folding cannot be random-walk.

Dill & Chan, 1997

Levinthal, 1968

Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


Objectives of this lecture9 l.jpg
Objectives of this Lecture

  • Overview Folding/Misfolding

  • Anfinsen

  • Levinthal Paradox

  • Folding Models

  • The denatured state

  • The molten globule

  • Two-state folding

  • Deciphering complex folding pathways

Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


The three protein folding models l.jpg

Oznur’s slide:

The Three Protein Folding Models

Framework

model

Hydrophobic collapse

model

Nucleation condensation

model

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

Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


Objectives of this lecture11 l.jpg
Objectives of this Lecture

  • Overview Folding/Misfolding

  • Anfinsen

  • Levinthal Paradox

  • Folding Models

  • The denatured state

  • The molten globule

  • Two-state folding

  • Deciphering complex folding pathways

Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


Random coil and denatured state l.jpg

Oznur’s slide:

Random Coil and Denatured State

Flory’s isolated pair hypothesis

Rg values determined by SAXS

“Φ,Ψ angles of each residue is sterically independent”

There should not exist any non-local interactions.

Rg values of 28 denatured proteins

obeys the Flory’s power law.

  • Rg= RgNv

  • N = Length (Residues)

  • v = Solvent viscosity parameter

  • Sosnick, T.R., et al. 2004

    Flory, 1969.

    Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


    Testing the random coil statistics l.jpg

    Oznur’s slide:

    Testing the random coil statistics

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

    33 proteins

    Number of residues

    Simulated Rg follows the power law.

    Despite 92% of the native structure kept,

    random coil statistics are obtained.

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

    Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


    The denatured state does flory s hypothesis hold l.jpg

    Oznur’s slide:

    The Denatured StateDoes Flory’s hypothesis hold?

    Conformations of polyalanine chains are enumerated to test the hypothesis.

    + ={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.

    Pappu et.al 2003.

    Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


    Which nmr spectrum is of folded and which is of unfolded lysozyme l.jpg
    Which NMR spectrum is of folded and which is of unfolded lysozyme?

    Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


    Which nmr spectrum is of folded and which is of unfolded lysozyme16 l.jpg
    Which NMR spectrum is of folded and which is of unfolded lysozyme?

    folded

    unfolded

    Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


    How would you use nmr to test for residual structure l.jpg
    How would you use NMR to test for residual structure? lysozyme?

    Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


    How would you identify residual structure in unfolded proteins with nmr l.jpg
    How would you identify residual structure in unfolded proteins with NMR?

    • What types of NMR parameters do you know?

      • chemical shifts

      • coupling constants

      • HetNOE

      • longitudinal relaxation rates (R1)

      • transverse relaxation rates (R2)

    Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


    How would you identify residual structure in unfolded proteins with nmr19 l.jpg
    How would you identify residual structure in unfolded proteins with NMR?

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

    • chemical shifts

    • coupling constants

    • HetNOE

    • longitudinal relaxation rates (R1)

    • transverse relaxation rates (R2)

    • 2. Comparison of NMR parameters with random coil

    • 3. Deviation from random coil identifies residual structure

    • Application to unfolded conformations of hen egg white lysozyme:

    • oxidized in 8M urea

    • reduced and methylated in 8M urea

    • reduced and methylated in water

    Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


    Chemical shift differences between unfolded lysozyme and random coil l.jpg
    Chemical shift differences between unfolded lysozyme and random coil

    Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


    Dynamics in folded unfolded lysozyme l.jpg
    Dynamics in folded/unfolded lysozyme random coil

    Unfolded:

    Arrows indicate oxidized (all disulfide bonds present) lysozyme

    Folded:

    Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


    Relaxation rates in unfolded lysozyme l.jpg
    Relaxation Rates in Unfolded Lysozyme random coil

    Unfolded lysozyme can be studied in 8 M urea.

    Unfolded lysozyme can also be studied without urea, if the disulfide bonds are reduced and the cysteines are derivatized to prevent them from forming disulfide bonds.

    Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


    Relaxation rates in unfolded lysozyme23 l.jpg
    Relaxation Rates in Unfolded Lysozyme random coil

    What do you observe?

    Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


    Relaxation rates in unfolded lysozyme24 l.jpg
    Relaxation Rates in Unfolded Lysozyme random coil

    Regions with higher relaxation rates are localized as clusters.

     Presence of clusters of residual structure that are restricted in conformational space, thus relax faster.

    Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


    How would you analyze the relaxation data l.jpg
    How would you analyze the relaxation data? random coil

    Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


    What are the assumptions of the model free approach l.jpg
    What are the assumptions of the model-free approach? random coil

    Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


    Analysis of the relaxation data l.jpg
    Analysis of the relaxation data random coil

    Three means of analysis have been proposed:

    • Model-free approach

    • Cole-Cole distributions

    • Gaussian clusters

    However: What gives rise to these clusters is not known.

    Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


    Relaxation rates in unfolded lysozyme28 l.jpg

    3. random coil

    2.

    5.

    4.

    6.

    1.

    Random Coil Model of Segmental Motion

    + Gaussian Distributions of Deviations

    2

    -

    -

    |

    i

    x

    |

    |

    i

    j

    |

    N

    0

    -

    -

    å

    å

    =

    +

    l

    R

    (

    i

    )

    R

    e

    Ae

    b

    int

    rinsic

    =

    j

    1

    x

    0

    Relaxation Rates in Unfolded Lysozyme

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

    Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


    Mapping of residual structure on the native structure l.jpg
    Mapping of residual structure on the native structure random coil

    Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


    Hydrophobic clusters of residual structure l.jpg
    Hydrophobic clusters of residual structure random coil

    Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


    What stabilizes the clusters of residual structure l.jpg
    What stabilizes the clusters of residual structure? random coil

    Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


    What stabilizes the clusters of residual structure32 l.jpg
    What stabilizes the clusters of residual structure? random coil

    • Long-range interactions?

    • Local structure?

    • How would you test this?

    Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


    Approach 1 l.jpg
    Approach 1 random coil

    • Peptides: if peptides without structural context of the full chain contain structure, then this structure is independent of long-range stabilization

    Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


    Approach 2 l.jpg
    Approach 2 random coil

    • Test for the presence of long-range interactions in the context of the full-length protein

    • What approaches can you imagine to test for long-range interactions?

    Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


    Residual structure mapped onto native structure l.jpg
    Residual Structure Mapped onto Native Structure random coil

    Clusters of deviations from random coil dynamics map onto proximal regions in the native structure, except cluster 3.

    Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


    How would you test for the presence of long range interactions approach 1 study effect of mutation l.jpg
    How would you test for the presence of long-range interactions?Approach 1. Study effect of mutation

    Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


    Effect of mutation on chemical shifts l.jpg
    Effect of mutation on chemical shifts interactions?

    Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


    Effect of mutation on relaxation rates l.jpg
    Effect of mutation on relaxation rates interactions?

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

    Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


    Effect of mutation on chemical shifts39 l.jpg
    Effect of mutation on chemical shifts interactions?

    Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


    Effect of mutation on relaxation rates40 l.jpg
    Effect of mutation on relaxation rates interactions?

    Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


    Model for unfolded ensemble l.jpg
    Model for unfolded ensemble interactions?

    Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


    Compactness by nmr l.jpg
    Compactness by NMR interactions?

    Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


    Approach 2 fret l.jpg
    Approach 2. FRET interactions?

    - So far has been only used for global changes, not to detect specific contact formation

    Haustein and Schwille (2004) Current Opin. Structural Biology 14, 531-540.

    Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


    Approach 3 epr proton relaxation l.jpg
    Approach 3. EPR – proton relaxation interactions?

    interaction up to 20-25Å

    Staphylococcus nuclease – Gillespie and Shortle (1997) JMB 268, 170-184 and 158-169.

    Molecular Biophysics III – Klein-Seetharaman – Folding Lecture


    Role of disulfide bonds for dynamics l.jpg

    A. Wild-Type interactions?

    B. W62G

    Role of disulfide bonds for dynamics

    Disulfide bonds and hydrophobic clusters are cooperative.

    Molecular Biophysics III – Klein-Seetharaman – Folding Lecture