-Thermodynamics & Reaction Engineering

-Unfolding of protein due to temperature

Dr. Chinmaya Mahapatra,[Link],PhD

Assistant Professor

Department of Biotechnology
National Institute of
Technology
Raipur-492 010 ,Chhattisgarh
Basic protein structure and stability :
Thermodynamics of protein stability
 Native and denatured states
denatured ensemble native state
unfolded ensemble folded state

many different structures single structure or

fluctuating; ensemble
not usually very compact; of very similar structures;
disordered but not a “random coil” compact
For some proteins, but not all, this process is readily reversible and
occurs
without populated intermediate forms--> “two-state” folding
 Naive view of folding thermodynamics
protein becomes less stable at
ΔGu high temp and unfolds when TΔS
Native Denatured exceeds ΔH
(folded) (unfolded)

ΔGu = ΔHu - TΔSu

ΔGu
ΔH
0
+ +
TΔS
u
u
favorable native state unfolded state
interactions broken more disordered
T
 Less naive thermodynamics of unfolding
Free energy of unfolding actually varies in a more complicated way with T.
Enthalpy and entropy are both temperature dependent.
Temperature dependence is described by the heat capacity ΔCp.

ΔHu = ΔHu0 + ΔCp (T–T0 ) enthalpy and entropy not

temperature independent

ΔSu = ΔSu0 + ΔCp ln (T/ T0 )

ΔGu = ΔHu0 – TΔSu0 + ΔCp[T– T0 –T ln (T/ T0 )]

figures into the total free energy as this term

T0 is some arbitrary reference temperature, and ΔHu0 and ΔSu0 are the
enthalpy and entropy at this temperature.
from Becktel & Schellman,
Biopolymers 26, 1859
Thermodynamic breakdown of unfolding
variation in enthalpy, entropy
huge compared to free
energy
TΔSu
ΔH
slope = u
ΔCp this example:
ΔHu, 298 = +35 kcal mol-1
ΔSu, 298 = +100 cal
mol-1K-1
ΔCp = +1500 cal mol-1K-1

ΔGu
ΔCp typically 10-18 cal
mol-1K-1 per residue (large
and positive)
 Temperature of maximal stability
point of maximal stability
occurs when TΔS is zero (Ts)

below Ts, entropy favors folding: folding

less favorable as decrease temp

this line
represents
zero above Ts, entropy disfavors folding:
folding less favorable
as increase temp

from Becktel & Schellman,

Biopolymers 26, 1859
(1987).
 Stability curves for proteins
Tc --> cold denaturation
temperature--usually below freezing proteins typically not very
stable:
5-20 kcal/mol at room temp
proteins typically have their
maximum stability near room
temp

Tm of a protein --> temperature

at which folded/unfolded states
equally populated--> ΔGu = 0

proteins with higher

heat capacity have A protein with a higher
tighter, steeper room
parabolas (red curve vs. temp stability could, in
blue) principle, have a lower Tm.
 Equation for thermal denaturation

ΔGu = ΔHu0 – TΔSu0 + ΔCp[T– T0 –T ln (T/ T0 )]

assign Tm as the arbitrary reference
temperature T0

ΔGu = ΔHu,Tm – TΔSu,Tm

+ ΔCp[T– Tm –T ln (T/ Tm)]
ΔGu, Tm = 0 ΔHu,Tm = Tm
so ΔSu,Tm
ΔGu = ΔHu,Tm(1 – T/ Tm )
+ ΔCp[T– Tm –T ln (T/ Tm)]
Amount of unfolded
protein as function
of T eqn describing ΔGu
as function of T

ΔGu = ΔHu,Tm(1 – T/ Tm )
+ ΔCp[T– Tm –T ln (T/ Tm)]

Ku = exp(−ΔGu/RT) = [U]/[F]
Keq for unfolding reaction
concentration unfolded
and folded
fu = Ku / (1 + Ku )
fraction unfolded set of nested equations
 Heat denaturation curve
so if I can somehow measure the
basic sigmoidal
folding transition... shape of this curve
derives from the
“two-state” nature of
the transition, but its
specific shape will
I can in principle extract vary with ΔCp, ΔH
the ΔCp, ΔH and the Tm
by fitting the curve Tm

and also get ΔG at every

temperature.
 Heat capacity and surface area

from Myers et al. Protein Sci 31, 2138 (1995)

Empirical studies of denaturation of proteins of known structure show that

ΔCp of unfolding (y-axis) depends on the ΔASA (change in accessible
surface area) upon folding (in other words the amount of surface buried).
Note that proteins with disulfide (open circles) fall below the curve...why?
 from Myers et al. Protein Sci 31, 2138 (1995)

...and as we have seen the ΔASA depends upon the size of the protein,
in terms of the number of residues in the polypeptide chain. This means that
ΔCp will be fairly predictable for globular proteins of a given size...on average,
it’s about 14 cal/(mol-K-residue), but it can be as low as 10 or as high as 18.
Liquid hydrocarbon model for heat capacity

• The dependence of heat capacity of unfolding upon surface area

burial suggests that it might be explained simply as a function of
burying the chemical groups in the protein side chains and/or main
chain.

• Indeed, it has been shown that a heat capacity change that parallels
that observed upon protein unfolding also occurs upon dissolution of
nonpolar solutes in water, so a major contributor may simply be the
burial of nonpolar groups--this is called the liquid hydrocarbon
model, which essentially explains the heat capacity in terms of the
resemblance of a protein interior to an oil drop.

• However, burial of the amide groups in the backbone also has an

effect on the heat capacity, based on experiments involving
dissolution of organic amides in water. It is smaller and opposite in
direction to the effect of burying hydrocarbons.
 Heat capacity and burial of surface
ΔCp = 0.32* ΔASAnp - 0.14* ΔASApol
plot showing ΔASAnp and
ΔASAp for a dozen proteins of
different size
based on dissolution based on dissolution of amide
of hydrocarbon compound solutes in
solutes in water water--note
is opposite in sign.
The relationship above does a pretty
good job of predicting heat capacities
of unfolding just by treating the protein
as a collection of nonpolar and polar
solutes. The nonpolar surface area
burial is the dominant effect and
determines the sign of the heat capacity
effect, both because the coefficient is
larger and because more nonpolar than
polar surface is buried when proteins
fold. from Spolar et al. Biochemistry 31, 3947
(1992)
 Chemical denaturants

urea
guanidine (guanidinium)

Molecular dynamics simulations of urea

denaturation suggest that it denatures
proteins by several mechanisms: stronger denaturant than urea
--competes for backbone hydrogen also a salt, unlike urea
bonds.
--some effect on solvation of
hydrophobic core
--affects dynamics/structure of water, See Bennion & Daggett, PNAS 100, 5142 (2003).
altering the hydrophobic effect
Chemical denaturation curve

Cm

fraction unfolded in the transition zone

can be translated into ΔGu values at each
urea concentration--> see next slide
Linear extrapolation to zero / m value
both guanidine & urea melt
should extrapolate to same
value of ΔGu H2O here about 4 kcal/mol

ΔGu = ΔGu H2O + m [denaturant]

urea
m is the slope of the
ΔGu vs. [denaturant]
curve: for urea here,
guanidine
it is 1.8 kcal mol-1 M-1

Data are ΔGu values

extracted from fu in
transition zone of
melt
 Stability curves determined from melts
pay no
attention from transition zones
to this of thermal melts
scale--
7 here is
equiv. to
zero.
from chemical
denaturation at 3
different temps

from Bowie & Sauer Biochemistry 1989, 28,

7139.
 m values correlate with surface area
burial, just like ΔCp
from Myers et al. Protein Sci 31, 2138 (1995)

notice how proteins with disulfide crosslinks (open circles) fall

below the line...the authors corrected for this and ultimately
came up with the following equation:
m (urea) = 0.14 * (ΔASA – 995*# crosslinks)
Proteostasis
The maintenance of an active set
of cellular proteins required under
a given set of conditions is called
proteostasis (Fig. 4-25). Three
kinds of processes contribute to
proteostasis. First, proteins are
synthesized on a ribosome.
Second, multiple pathways
contribute to protein folding, many
of which involve the activity of
complexes called chaperones.
Chaperones (including chaperonins)
also contribute to the refolding of
proteins that are partially and
transiently unfolded. Finally,
proteins that are irreversibly unfolded are subject to sequestration
and degradation by several additional pathways. Partially unfolded
proteins and protein-folding intermediates that escape the quality
control activities of the chaperones and degradative pathways may
aggregate, forming both disordered aggregates and ordered
amyloid-like aggregates that contribute to disease processes.
 Protein Denaturation (I)
The conformation of a native protein is
only marginally stable. Conditions
different from those in a cell, etc.,
therefore can cause protein structural
changes resulting in the loss of
three-dimensional structure needed for
function (i.e., denaturation). In the
denatured state, the conformation of the
protein need not be completely
randomized. A number of physical and
chemical agents can cause protein
denaturation. A classic agent is heat,
which has complex effects on many weak
interactions in a protein (primarily on the
hydrogen bonds). On heating, a protein’s
conformation generally remains intact until
an abrupt loss of structure occurs over a
relatively narrow temperature range (the
Tm) (Fig. 4-26a). The abruptness of the
loss of structure suggests a cooperative
process in which loss of structure in one
or more parts of the protein rapidly
destabilizes the structure of other parts.
 Protein Denaturation (II)
Proteins also can be denatured by
extremes in pH, by miscible organic
solvents such as alcohol and acetone, by
certain solutes such as urea and guanidine
hydrochloride, or by detergents (Fig.
4-6b). None of these agents breaks
covalent bonds. Organic solvents, urea
and detergents act primarily by disrupting
the hydrophobic interactions that produce
the stable core of globular proteins. Urea
and guanidine hydrochloride also disrupt
hydrogen bonds. Extremes of pH alter the
net charge on the protein causing
electrostatic repulsion and the disruption
of some hydrogen bonding. The denatured
structures resulting from these various
treatments are not necessarily the same.
Lastly, denaturation often results in
aggregation and precipitation of the
unfolded protein. The protein precipitate
that is seen after boiling an egg white is
a well known example.
 Key points about protein stability

• in general protein native states are weakly stable

(5-20 kcal/mol) relative to unfolded states
• they tend to be maximally stable around room
temperature, and are subject to both cold and heat
denaturation, with inversion of sign of both the
enthalpy and the entropy of unfolding
• large heat capacity change due partly to properties
of water--large T dependence of enthalpy, entropy
• much of the denaturation behavior of proteins can
be understood in terms of simple burial and solvent
exposure of nonpolar and polar surface area
Thank You