The twostate model of protein unfolding

1
The two-state model of protein unfolding
Observation same transition curve with different
methods fluorescence (buried Trp) circular
dichroism (secondary structure) near-UV CD
(buried aromatic groups) enzyme activity
(active site) NMR (environment of individual
hydrogen atoms) Strong qualitative evidence
But A quantitative thermodynamic test is
available (see later)

• it is an approximation
• strictly limited to reversible transitions
• not true for many proteins

1
2
The two-state model of protein unfolding
x is the unfolding coordinate (T, pH,
denaturant,) B is the observed property (B is
extrapolated into the transition region)
Ready access to thermodynamic functions Precision
only if 0.02 lt fu lt 0.98 I.e., when -2.3 lt
DGunf lt 2.3 kcal/mol
2
3
Protein unfolding solvent denaturation
Protein denaturants Urea (OC(NH2)2) Guanidine
(C?(NH2)3) Ethanol
3
4
Protein unfolding solvent denaturation
Linear dependence, slope independent of
T, (Tanford) Extrapolation gives DGFU at zero
denaturant
50 40 25 data
2.3 kcal/mol 0 2.3
No data outside this zone
-DGFU
2 4 6 M Gu
4
5
Solvent denaturation and preferential
interactions with different solvent components
Many (M) very weak sites
Linear dependence of ln K on U
5
6
Thermal denaturation
Same experiments, replotted
Apply the vant Hoff equation to obtain DH and
DCP DH depends on T DCP is (approx.) independent
of T
6
7
Thermal denaturation
Composite result
Typical values!
7
8
Cold denaturation
Because DCp is constant, DH becomes lt 0 at low
T. Typically, this occurs at or blow 0C
Observe in Super-cooled solution (Wands
micro-drops) Mutants
T4 lysozyme mutant From Nelson et al., J. Am.
Chem. Soc. 123, 7453-7454, 2001)
8
9
Calorimetry
Directly measure Cp H ? Cp dT S ? (Cp /T)
dT G/T ? (H /T) dT Apply to Cp and ?Cp,FU
Can estimate fu
Measurement is per gram Previous estimates are
per mol Ratio is the size of the cooperative unit
9
10
Near-native unfolding H isotope exchange
Hydrogen isotope exchange with solvent H bonded
to C no exchange H bonded to O very fast H
bonded to N base catalyzed, slow at pH 3
H to D exchange Measure density of solvent
(global old method) Measure NMR line intensity
(local)
10
11
Near-native unfolding not two-state
Hydrogen isotope exchange with solvent 1.
Exchange rate (local) exposure ? free exchange
rate kx fu ? kx,0 (slow intrinsic
exchange) 2. Exchange rate rate of
unfolding kx ku (fast intrinsic exchange)
Exposure varies over many orders of
magnitude fu ? fu,global Identify core
structures (nmr)
Rates converge as T approaches transition
midpoint fu ? fu,global near Ttr
11
12
Protein stability not a maximum
Thermophiles and hyperthermophiles Engineered
hyperstable mutants Very stable designed proteins
Why? accident high stability ? fewer
sequences design proteins must be
degradable function need flexibility folding
need unique stable structure
12
13
The molten globule
Occurs rarely dominant state of a native
protein first intermediate in folding (from
highly unfolded) (carelessly) designed
proteins Properties intermediate helix or beta
content - some tertiary contacts (perhaps
non-native) non-cooperative further
unfolding few NOE peaks binds ANS
13
14
The molten globule
A third state unfolded ? gas folded ?
crystal ?? ? liquid Cf. a simple phase diagram
No equivalent of P May not ever see (b)
14
15
A very nice book
Finkelstein Ptitsyn, Protein Physics, Academic
Press, 2002 80 on amazon
15
16
Thermodynamic parameters for protein unfolding

• a. Factors that contribute to protein
  conformational stability (DG)
• Hydrophobic interactions
• Intramolecular hydrogen bonds
• Intramolecular van der Waals forces (good
  packing)
• Intramolecular salt bridges (pairs of ionic
  groups)
• b. Factors that contribute to protein
  conformational instability
• Hydrogen bonds with solvent
• van der Waals interactions between protein and
  solvent
• Solvation of ionic groups
• Conformational freedom

16
17
Thermodynamic parameters
Factors that contribute to enthalpy (DHFUgt0)
hydrogen bonds van der Waals forces
Factors that contribute to entropy
(DSFUgt0) hydrophobic interactions conformational
freedom
Factors that contribute to specific heat (DCpgt0)
hydrophobic interactions
17
18
Stabilization of various structures by
interaction of apolar parts
Protein molecules Micelles Lipid bilayers
hydrophobic stabilization poor mixing of
hydrocarbons and water
18
19
Hydrophobic interactions (1)
cf. High surface free energy (surface pressure)
of water
It costs energy to make a hole in
water Concept It costs free energy to make a
molecule-sized hole in water (DGgt0)
Non-polar side chains are exposed in unfolded
state, buried in folded state, and this
stabilizes the folded state Kauzman Tanford
19
20
Hydrophobic interactions (2)
High hydrogen-bond content of liquid water Low
energy of liquid water (also low entropy)
Concept Relate the energy changes to changes in
hydrogen bonding in water, when transfer an
apolar solute molecule to water Less hydrogen
bonding DHtr gt 0 (More hydrogen bonding DStr lt
0)
20
21
Surface free energy
Planar water-air interface at room temp DG
100 cal/(mol Å2) DH 175 cal/(mol Å2) TDS 75
cal/(mol Å2) Dcp ? 0 cal/(molÅ2degree)
Structure is more free Hydrogen bonds have been
lost
21
22
Transfer free energy
Methane into water DG 2600 cal/mol DH
2700 cal/mol TDS 5300 cal/mol Dcp ?
C6H12 into water DG 2300 cal/mol DH 7300
cal/mol TDS 9600 cal/mol Dcp 94
cal/(mol?degree)
Structure is less free Hydrogen bonds have been
gained
Large DCp for protein unfolding implies that
methane-water (not air-water) is the appropriate
model
Pictures are premature Simulations reproduce DGgt0
but not DHlt0
22
23
Transfer free energies a hydrophobic scale for
the amino acids
Amino acid R-H
DGtr Pro (see note) 2.48 Gly hydrogen 2.39 Leu is
obutane 2.28 Ile n-butane 2.15 Val propane 1.99 Al
a methane 1.94 Phe toluene 0.76 Cys methanethiol
1.24 Met methyl ethyl sulfide 1.48 Thr ethanol
4.88 Ser methanol 5.06 Trp 3-methylindole 5.88 T
yr 4-cresol 6.11 Gln propionamide 9.38 Lys n-but
ylamine 9.52 Asn acetamide 9.68 Glu propionic
acid 10.20 His 4-methylimidazole 10.27 Asp aceti
c acid 10.95 Arg n-propylguanidine 19.92
kcal/mol
Wolfenden
23