Fundamentals and Thermodynamics of Protein Folding

1
Fundamentals and Thermodynamics of Protein Folding

• Richard Thompson
• Dept of Biochemistry and Molecular Biology
• GPILS 709 Advanced Biochemistry
• Spring 2007

2
Outline

• Basics of 2, 3 protein structure
• Thermodynamics of noncovalent interactions
• electrostatic, hydrogen bonding, hydrophobic
• Overview of the protein folding problem
• Conceptual, experimental, computational issues
• Practical aspects of folding disease,
  biotechnology
• Issues Pathway, Kinetics
• Approaches
• Fundamental Observe denature/renature
• Spectroscopies
• Computational

3
1, 2 ,and 3 Protein Structure
Fig. 3-16 from Lehninger Biochemistry, 4th ed.
4
Weak, Noncovalent Interactions

• 2 , 3 Structure supported by weak, noncovalent
  forces
• Net stability of folded protein is lt10 kcal/mol
• Sum of many weak interactions
• Strongly condition dependent temp, pH, ionic
  strength
• Most ?G from hydrophobic interaction others net
  0
• Thermodynamics includes solvent water
• Disulfides intermediate in strength

5
Hydrogen Bonding

• Shared H atom between N, O sometimes Cl, F
• H X bond aligned with unpaired electrons
  strongly directional
• Intramolecular H-bond vs. with solvent water
• Figure 2-11 from Lehninger Biochemistry, 4th Ed.

6
Electrostatic Bonds Salt Bridges

• Unlike charges attract
• Coulombic interactions weak in polar solvent
• Competition between ion pairing and solvated
  charges
• Energetically unfavorable in hydrophobic interior
• Enthalpy predictable from coulomb theory

7
Ions are Happier Hydrated Salt Dissolves in Water
Figure 2-6 from Lehninger Biochemistry, 4th Ed.
8
Enthalpy of Ion Pair Formation
Enthalpy of forming ion pairs (in vacuo) is
closely approximated by self-charging of spheres
of atomic size. In water, ion pairing vs.
solvated, separate ions is ?G 0 Figure 5-9
from Kyte, Structure in Protein Chemistry
9
Free Energy of Ion Pair Formation
10
Hydrophobic Interactions Entropy-Driven

• Main thermodynamic source of protein stability
• The water doesnt dislike grease, it just likes
  itself more
• Hydrogen-bonded clathrate cage minimize surface
  area
• Hydrophobic effect maximizes entropy of the
  water Strongly temperature-dependent

11
Water Forms a Transient, H-Bonded Lattice
Water molecules are H-bond donors,
acceptors Form H-bonded lattice Maximize config.
entropy by having many partners Non H-bonding
solute in cavity water lining cavity has low
entropy, so minimize surface area
12
Hydrophobic Effect is Entropy Driven (1)
Entropy plotted vs. surface area of cavity
enclosing hydrophobic solute in water smaller
molecules require smaller cavity, reduce entropy
less, are more soluble Upper panel aliphatic,
lower panel aromatic Figure5-21 from Kyte
13
Hydrophobic Effect is Entropy Driven (2)
Free energy of transferring benzene from water to
benzene as a function of temperature. Note
relative contributions of ?S and ?H to ?G, and
change with temperature Figure 5-19 from Kyte
14
Hydrophobic Residues on the Inside
Hydrophobic residues and heme are hidden from the
outside most ?G stabilizing protein comes from
hydrophobicity
15
Cysteine Disulfides A Reversible Covalent Bond
16
Must have correct disulfide formation during
folding Secreted proteins often have disulfides
to stabilize extracellularly in oxidizing
environment Reduction promotes denaturation
break crosslinks
17
Protein Secondary and Tertiary Structure

• Peptide bond stereochemistry
• Peptide bond is usually planar
• Ramachandran/Sasisekharan diagram
• Secondary structural motifs
• Alpha helix other helices
• Beta pleated sheets
• Tertiary structural motifs
• Leucine zipper, zinc finger, helix-turn-helix

18
Peptide Bond is Planar
C-N has double bond character Ca-N-C-Ca are
coplanar Polypeptide path determined by ?(Ca-C)
and F (N-Ca) angles
19
Ramachandran Diagram
Blue areas show values of F, ? angles where
steric hindrance is minimum values plotted for
protein of known structure mostly fall in
blue Exceptions are Gly (R group is H) and
Pro Chap.4, Lehninger, 4th Ed.
20
Alpha Helix (1)
Chain twists into a helix with 36 residues per 10
turns (3.6 residue/turn) Chain COs make
straight H-bonds with N-Hs 3 residues away
H-bonds stabilize structure Pro often breaks helix
21
Alpha Helix (2)
a-helix R-groups stick out radially, dont
interfere sterically. Helix is not very
flexible Center is not hollow
22
Collagen Triple Helix
Collagen helix has sequence Gly-X-HyPro 3
strands twist together like rope
23
Other Helices 310 and p Helices
310 (left) and p (right) helices have fewer
H-bonds, less straight than alpha helix are less
stable Seldom seen in protein structure
24
Beta-Pleated Sheet (1)
Antiparallel means each strand goes opposite
direction N?C from its neighbor strands may be
connected by ß-turns R-groups on opposite sides
of sheet Sheet may be twisted
25
Beta-Pleated Sheet (2)
Parallel sheets are less stable, less common than
antiparallel H-bonds fewer, less straight than
antiparallel ß-sheet ß-sheet is more flexible
bend into cylinders, saddles
26
Secondary Structural Motifs have Discrete
Positions on the Ramachandran Diagram
Amino acids in right-handed a-helices will have F
(-50), ? (-60) Other motifs have different
F, ? values
27
Tertiary Motifs Leucine Zipper
Leu zipper appears in sequence with Leu every 7
th residue Note twisting of a-helices Figure
28-14 of Lehninger Biochemistry, 3rd Ed.
28
Tertiary Motifs Helix-Turn-Helix
Helix-Turn-Helix motif binds in major groove of
DNA protein helix makes contacts with edges of
DNA bases recognizes DNA sequence Figure 28-11
of Lehninger Biochemistry, 3rd. Ed.
29
Tertiary Motifs Zinc Fingers and Friends
Cys, His residues in a-helix, ß-turn hold Zn ions
rigidly contacts with DNA bases permit
recognition of DNA sequence Hundreds of examples
known from Cys, His-rich gene sequences Figure
28-12 in Lehninger Biochemistry 3rd Ed.
30
Canonical Folds 4-Helix Bundle
4-Helix bundle seen in diverse proteins
myohemerythrin, human growth hormone, cytochrome
b562. Basic fold used in semisynthetic
proteins Fig1-51 from Petsko and Ringe, Protein
Structure and Function
31
Canonical Folds Beta-Barrel
32
Protein Folding Overview

• Expressed polyamino acid rapidly folds into
  compact, active, (semi-)unique conformation

Fig 4-28 from Lehninger Biochemistry, 4th ed.
33
Practical Aspects of Protein Folding

• Diseases of protein misfolding (amyloidoses)
• Spongiform encephalopathies (Mad cow,
  Creutzfeldt-Jakob)
• Alzheimers Disease
• Cystic Fibrosis
• Production of expressed proteins (biotechnology)
• Inclusion bodies in bacteria

34
Protein Folding Conceptual Issues

• Leventhals paradox (1969) too many conformers
  to search through
• Ill-defined starting and end-point
• All information in sequence (C. B. Anfinsen)
• Competition between folding and aggregation
• Sometimes have help chaperones

35
Chaperones
Chaperones (GroEL, hsp70) enclose nascent
protein, provide isolation to prevent aggregation
before fold completed Figure 4-31b from
Lehninger Biochemistry, 4th ed.
36
Protein Folding Experimental Issues

• Rapid process µsec to msec
• Ensemble averages of different states, pathways
• Minimize intermolecular contacts low protein
• Complex systems hundreds of residues, contacts
• Main approach spectroscopy of folding

37
Spectroscopic Approaches

• Circular Dichroism
• General, insensitive, low resolution, slow (msec)
• Fluorescence
• Fast (psec), sensitive, low resolution, few
  fluorophores
• NMR
• High resolution, medium speed (msec) and
  sensitivity
• Mass spectroscopy
• Not fast, limited scope (H-D exchange), good
  resolution

38
Other Important Techniques

• H-D Exchange
• Measure with mass spec or NMR molecular detail
• Calorimetry
• Gives thermodynamics, no structural info slow
• Protein Engineering
• Replace individual residues and measure effects

39
Circular Dichroism
Left panel shows predicted and measured CD
spectra for poly-Ala right panel shows a-helix,
ß-sheet, random coil spectra
40
Hydrogen-Deuterium Exchange

• Exposed hydrogen-X (XO, N) exchanges rapidly
  with deuterium in D2O
• Degree of protection (P) from exchange during
  folding is measure of exposure
• Measure by NMR, mass spec

41
Cytochrome c H-D Exchange Protection
Protection of amide protons from exchange with
deuterons during early (2 msec) Cytochrome c
refolding in 0.3 M GuaHCl. Protected residues
(Pgt3) are darker in the structure and have higher
bars in the sequence only a few residues in the
N-helix, C-helix, and 60s are protected Sauder
and Roder (1998) Fold. Des. 3, 293.
42
Effect of Mutation on Folding Rates and
Equilibria (1)
Mutants of chymotrypsin inhibitor 2 (?,?) unfold
at lower GuaHCl than wild type (?) are
generally less stable. Express residue
contribution to stability as F 0 (not involved)
to F 1 (highly involved) Map residues most
important to folding, stability
43
Effect of Mutations on Folding Rates, Equilibria
(2)
Mutations can also affect folding rates, by
changing activation energy (?G ) F ??G-D /
??GN-D expresses contribution of residue to
folding activation energy, and thus folding
rate From Fig 18-10 of Fersht
F 0 F 1
44
Main Approach Spectroscopy of Folding/Unfolding

• Denature protein by temperature jump, denaturant
• Renature predenatured protein dilute denaturant
• Stopped flow msec kinetics
• Continuous flow faster but lots of material
• Fluorescence, CD, NMR, Mass spec (H-D exchange)

45
Folding Kinetics by Fluorescence Measurement
Fluorescence of Trp-59 drops when close to heme
in folded cytochrome c due to energy
transfer µsec processes by continuous flow, other
by stopped flow Shastry and Roder (1998), Nature
Struc. Biol. 5, 385.
46
Protein Folding Computational Approaches

• A major problem in molecular dynamics simulation
• Thousands of atoms interacting for 106 psec
• Use simple systems BPTI
• Need psec resolution to verify by experiment
• Lattice models helpful (K. Dill)

47
X-Pro cis-trans Isomerization

• Peptide bonds rarely ( 0.1 ) in cis form
• Cis X-Pro more common ( 10 )
• Cis-trans isomerize slowly (sec min) ?H 20
  kcal
• Often rate-determining in folding process
• Can be enzyme catalyzed
• Cyclophilin, FK506 binding proteins, parvulins

48
Disulfide Bond Formation

• Secreted proteins, many others have disulfides
• Disulfides form by oxidation, often by disulfides
• Disulfides reduced by cellular thiols
• Correct pairing of gt2 cys essential
• Protein disulfide isomerases catalyze
  interconversion

49
Mechanism / Pathway of Folding

• Some defined intermediates Barnase, lysozyme
• Detailed pathways not yet observed
• Folding funnel model
• Molten globule
• Many small proteins are one step D ? N

50
Proposed Folding Pathways
Figure from Fersht
51
Folding Funnel
As helices form and hydrophobic collapse occurs,
protein goes through molten globule states with
some 2, but non-native 3 structures. Globule
states go to discrete intermediates, which
overcome activation barrier(s) to get to final,
native conformation Figure 4-29 from
Lehninger Biochemistry, 4th ed.
52
Molten Globule States

• Some folding intermediates have molten globule
  structure
• Substantial 2 structure, little 3 structure
• Collapsed state but disordered core accessible to
  solvent
• Obtained by acid denaturation
• Often binds fluorescent dye ANS

O. Ptitsyn, Adv. Prot. Chem. 47, 83 (1995)
53
Molten Globule State of a-Lactalbumin
GuaHCl-induced unfolding of a-lactalbumin
monitored by CD at 222 and 270 nm Disorder
appears in aromatic region at lower GuaHCl,
indicating molten globule state with some
secondary structure Ikeguchi, et al., (1986) J.
Biochem (Tokyo) 99, 1191.
54
Molten Globule State of a-Lactalbumin
CD spectra in the peptide range (200 240 nm)
show native-like 2 structure (except 6, 6N
GuaHCl), but acidic molten globule (3) in
aromatic wavelengths is similar to thermally
unfolded (s 4 and 5)
Figure from Kuwajima, et al., Biochem. 24, 874
(1985)
55
Single Step Folding
Many small proteins fold reversibly N ? D without
discernable intermediates
Ln kobs (net rate kfold kunfold ) plotted
vs. denaturant gives chevron plot
chymotrypsin inhibitor 2 fits two state model
56
Chevron Plots Identify Intermediates

• Chevron plot for barnase with lower kfold than
  expected for refolding indicate an intermediate

Fig 18.6 from Fersht
57
Temperature Dependence of Folding Rate Eyring
Plot
Temperature dependence of refolding (left) and
unfolding (right) rates plotted as ln(k/T) vs.
1/T can provide T?S, ?H, and ?Cp of
activation Fig 3 of Bieri and Kiefhaber in R.H.
Pain, ed., Mechanisms of Protein Folding, 2nd Ed.
58
Multiple Intermediates in Lysozyme Folding
Figure 6 from O. Bieri and T. Kiefhaber in
Mechanisms of Protein Folding, 2nd Ed. (R. H.
Pain, Ed.)