I investigated the topic too, so here is my answer.
To understand thermodinamic stability of water solved globulins or membrane proteins (all of them proteins hereafter) we have to understand protein folding. Proteins have a 3d structure (composed by their primary, secondary and tertiary structures). The structure of the proteins is constantly changing between the folded and unfolded states. The folded state has a lower free energy while the unfolded state has a higher. Between them there is a free energy barrier which determines the speed of the folding on a specific temperature.
Changing structural on environmental factors can affect these free energies and can shift the equilibrium to the unfolded state. The unfolded protein can suffer irreversible changes (aggregation, disulphide exchange, proteolysis, irreversible subunit dissociation, chemical degradation, etc...), so the denaturation of the protein can be reversible or irreversible.
Note that this is a very simplistic view, I think there are different degrees of unfoldedness, and so different things can happen when the protein is in one of these. For instance by a low degree of unfoldedness misfolding can happen which results a stable, but inactive folded state without coagulation (this can be more or less reversible). By a higher degree of unfoldedness coagulation can happen.
Folding mostly depends on one simple rule: all of the hydrogen bonds have to be satisfied, because a non-satisfied hydrogen bond has a very high energetic cost. The proteins have polar surfaces, which form hydrogen bonds with the water, and one or more apolar center, which have inner hydrogen bonds as backbones. Burial of an unpaired polar amino-acid (e.g. non-satisfied hydrogen bond) is very destabilizing and so it is non-existent in natural working proteins. Other factors, like salt bridges, aromatic-aromatic interactions, disulphide bonds, etc... can affect the stability as well, but hydrogen bonds and hydrophobic interactions are the major factors. The weights of these two major factors is most likely protein dependent (a study suggested 75% and 25%, while another 40% and 60%).
The backbone hydrogen bonds are usually most stable around room temperature, so the lowest free energy and the maximum stability is around 20°C by most proteins and both heating and cooling lowers the stability and can lead to denaturation. High temperatures (>80°C) can cause covalent degradation, and so irreversible denaturation. Pressure has similar effect on the hydrogen bonds and the stability as cold denaturation.
The osmolyte cosolvents like urea or TMAO contribute differently to the free energy of the folded and unfolded states and so shift the equilibrium between them. for example urea can cause denaturation, while TMAO protects the protein from denaturation. I think it is evident that changing pH and saltiness has strong effect on the charges of the amino acid side chains, and so the hydrogen bonds and the stability.
Both ultrasound and pulsed electric field (PEF) can cause denaturation. The effect seems strongly depending on the parameters of ultrasound/PEF and the type of the protein. Interesting that PEF can increase the enzyme activity too. It is hard to find studies about the denaturation mechanisms by these methods.
If we want to increase the protein stability, the method we choose can depend on what we want to protect the protein from. One or more methods from the following list can help to increase the stability:
- increase of compactness and better packing (minimalization of surface/volume ratio)
- increase of electrostatic interactions (formation of additional ion pairs, e.g. more glutamic acid)
- additional hydrogen bonds
- additional disulphide bridges
- increasing hydrophobic interactions (greater proportion of buried hydrophobic residues)
- change protein microenvironment (use osmolytes, change pH, saltiness)
- glycosylate the protein surface
- decrease chain length
- change surface amino-acids (the effect can be completely unpredictable, but there are surface residue patterns with known effect on stability; add more ionisable amino acids to the surface; bury hydrophobic residues; etc...)
- protein fixation can change the stability as well
Thermodynamic stability of a protein that unfolds and refolds rapidly,
reversibly, cooperatively, and with a simple, two-state mechanism. The
easiest proteins in which to study folding and stability are those
that exhibit this sort of rapid reversibility. Both experimental
design and also theoretical treatment of data are simplified by
reversible systems. Thus, it is no surprise that most of the
literature reports about stability discuss this type of reversible
system. The stability of the protein is simply the difference in Gibbs
free energy, dG, between the folded and the unfolded states. The only
factors affecting stability are the relative free energies of the
folded (Gf) and the unfolded (Gu) states. The larger and more positive
Gu, the more stable is the protein to denaturation.
If a protein unfolds reversibly it may be fully unfolded and inactive
at high temperatures, but once it cools to room temperature, it will
refold and fully recover activity. In the case of irreversible or
slowly unfolding proteins, it is kinetic stability or the rate of
unfolding that is important. A protein that is kinetically stable will
unfold more slowly than a kinetically unstable protein. In a
kinetically stable protein, a large free energy barrier to unfolding
is required and the factors affecting stability are the relative free
energies of the folded (Gf) and the transition state (Gts) for the
first committed step on the unfolding pathway. Irreversible loss of
protein folded structure is represented by: F <-> U -> I, where I is
inactive due to aggregation, disulphide exchange, proteolysis,
irreversible subunit dissociation, chemical degradation, etc...
Evidence from proteins and peptides supports the conclusion that
intrapeptide hydrogen bonds stabilize the folded form of proteins.
Paradoxically, evidence from small molecules supports the opposite
conclusion, that intrapeptide hydrogen bonds are less favorable than
peptide–water hydrogen bonds. A related issue—often lost in this
debate about comparing peptide–peptide to peptide– water hydrogen
bonds—involves the energetic cost of an unsatisfied hydrogen bond.
Here, experiment and theory agree that breaking a hydrogen bond costs
between 5 and 6 kcal/mol. Accordingly, the likelihood of finding an
unsatisfied hydrogen bond in a protein is insignificant. This
realization establishes a powerful rule for evaluating protein
In their description of the alpha-helix, Pauling et al. (1951)
asserted that the energy of the peptide N–H• • •O=C hydrogen bond was
of order -8 kcal/mol, and that “such instability would result from the
failure to form these bonds that we may be confident of their
presence.” Pauling’s earlier estimate of the total protein hydrogen
bond energy was -5 kcal/mol (Mirsky and Pauling 1936). From solution
studies of urea dimers, Schellman estimated that an intrapeptide
hydrogen bond would be enthalpically favored over a peptide–water
hydrogen bond by ~1.5 kcal/mol (Schellman 1955). These and similar
early studies led to the conclusion that the peptide hydrogen bond is
a significant factor in stabilizing protein conformations.
This view was to change dramatically following a famous review by
Kauzmann (1959), who invoked the thermodynamics of small model
compounds to argue that stabilization of the folded state of a protein
is due almost exclusively to the hydrophobic effect. Soon after
Kauzmann’s proposal, Klotz and Franzen (1962) determined that the
enthalpy of the interamide hydrogen bond of N-methyl acetamide in
water was zero, and concluded that “the intrinsic stability of
interpeptide hydrogen bonds in aqueous solution is small.” Similarly,
hydrogen bonding involving another small molecule,
epsilon-caprolactam, in dilute solution was shown to be negligible
(Susi and Ard 1969). Kauzmann’s proposal, bolstered by these later
studies, led to the widely held view that the hydrophobic effect makes
the major energetic contribution to protein stability, with hydrogen
bonds contributing little, or perhaps even opposing, the folding
process. See Baldwin (2003) for a recent discussion of these issues.
Protein hydrogen bonds are ubiquitous, directional, and largely local,
partitioning the polypeptide chain into alpha- and 3_10-helices,
beta-sheet, and beta-turns. Together, these hydrogen-bonded backbone
structures account for at least 75% of the conformation, on average,
with remaining residues participating in both additional
intramolecular hydrogen bonding and hydrogen bonding to water.
Unsatisfied backbone polar groups are energetically expensive, to the
degree that they almost never occur.
Force measurements between surfaces functionalized with lipids having
hydrogen bonding headgroups (NTA, A, T, and MeT lipids) lead to a
reproducible value of the energy of a single hydrogen bond in pure
water: ~0.5 kcal/mol. It shows that it is energetically more favorable
for the headgroups to make hydrogen bonds with each other than to make
hydrogen bonds with water molecules. This is coherent with past
studies made on proteins stability, which showed that intramolecular
hydrogen bonds in a folded protein are energetically more favorable
than bonds with water molecules in an unfolded protein with an average
stabilization of ~1 kcal/mol per intramolecular hydrogen bond.
The main determinant of cold denaturation tendency is likely the
stability decrease of backbone hydrogen bonds at low temperatures,
which in turn is affected by the packing manner of the hydrophobic
Using a newly developed pressure cell, we have now mapped pressure-
and temperature-dependent changes of 31 hydrogen bonds in ubiquitin by
measuring HBCs with very high precision. Short-range hydrogen bonds
are only moderately perturbed, but many hydrogen bonds with large
sequence separations (high contact order) show greater changes. In
contrast, other high-contact-order hydrogen bonds remain virtually
We study the stability of globular proteins as a function of
temperature and pressure through NPT simulations of a coarse-grained
model. We reproduce the elliptical stability of proteins and highlight
a unifying microscopic mechanism for pressure and cold denaturations.
The mechanism involves the solvation of nonpolar residues with a thin
layer of water. These solvated states have lower volume and lower
hydrogen-bond energy compared to other conformations of nonpolar
solutes. Hence, these solvated states are favorable at high pressure
and low temperature, and they facilitate protein unfolding under these
We further find that it is the changes in hydrophobic hydration with
decreasing temperature that drive cold unfolding and that the overall
process is enthalpically driven, whereas heat denaturation is
As a consequence of a weaker penetration upon pressurizing, it was
found that the pressure-denatured state was partially unfolded
compared with the heat-denatured state. The mechanism of pressure
denaturation was related to the disruption of the hydrogen-bond
network of water onto a set of clusters characterized by strengthened
O – H interactions, inducing a hardening of protein dynamics. The
mechanism is opposite to that observed upon heating, i.e., the
softening of the hydrogen bond network of water inducing a softer
We can conclude that the main driving force of protein denaturation at
high pressures is the decrease of the hydrophobic effect as a
consequence of the changes in water structure, without contradicting
any of the current theories on the hydrophobic effect.
For the pressure denaturation the weakening of the hydrophobic
interaction between the bulky side chains is found to be crucial at
lower temperatures, leading to an apparent destabilization of the
folded backbone structure at elevated pressures.
It is found that the energetics involving backbone hydrogen bonding
controls these shifts in protein stability almost entirely, with
osmolyte cosolvents simply dialing between solvent-backbone versus
backbone-backbone hydrogen bonds, as a function of solvent quality.
Overall, the sonication process had little effect on the structure of
proteins in WPC solutions which is critical to preserving functional
properties during the ultrasonic processing of whey protein based
The data presented here suggest that among proteins of fibrinolytic
systems, the fibrinogen is one of the most sensitive proteins to the
action of ultrasound. It has been shown in vitro that ultrasound
induced fibrinogen aggregates formation, characterized by the loss of
clotting ability and a greater rate of plasminolysis than native
fibrinogen in different model systems and under different mode of
The results obtained with the different experimental protocols
indicate, however, that the conformational equilibrium of GrpE is
insensitive to electromagnetic fields in the tested range of frequency
and field strength.
Effects of pulsed electric fields (PEF) treatment (0–547 µs and 0–40
kV/cm) on physicochemical properties of soybean protein isolates (SPI)
were studied. Solubility, surface free sulfhydryls (SHF) and
hydrophobicity of SPI dispersions (20 mg/ml) increased with the
increment of the PEF strength and treatment time at constant pulse
width 2 µs, pulse frequency 500 pulse per second (pps) and sample flow
rate (1 ml/s). When the PEF strength and treatment time were above 30
kV/cm and 288 µs, solubility, surface SHF, and hydrophobicity of SPI
decreased due to denaturation and aggregation of SPI by hydrophobic
interactions and disulfide bonds. Size-exclusion chromatography and
laser light scattering analyses demonstrated further that stronger PEF
treatment-induced dissociation, denaturation and reaggregation of SPI.
Circular dichroism analysis showed that PEF treatment did not produce
significant secondary structure changes of SPI.
A compact and low cost bench-top, pulsed electric field treatment
system was designed and developed. The unit consisted of a
high-voltage pulse generator (? 30 kV) and a treatment chamber with ?
148 ml capacity. Over the set-up voltage range of 4–26 kV, 30 pulses
(with instant charge reversal) were applied to eight different enzyme
solutions using a 0.3-cm electrode distance, a 13–87 kV/cm field,
0.5-Hz pulse frequency, 2-µs pulse width and 20 °C process temperature. For some enzymes, activities were reduced after the pulse
treatments: lipase, glucose oxidase and heat-stable ?-amylase
exhibited a vast reduction of 70–85%; peroxidase and polyphenol
oxidase showed a moderate 30–40% reduction whereas alkaline
phosphatase only displayed a slight 5% reduction under the conditions
employed. On the other hand, the enzyme activities of lysozyme and
pepsin were increased under a certain range of voltages. Electric
pulse profile (instant charge reversal) played a very important role
in reducing the activities of various enzymes.
Effects of high-voltage pulsed electric field (PEF) on native or
thermal denatured enzyme activities were studied. When PEF was applied
to various native enzymes, 105–120% of initial enzyme activities were
observed after PEF treatment. It was suggested that an activation of
enzyme would be possible by PEF treatment. We attempted a refolding of
thermal denatured enzyme by using PEF. When PEF was applied to
denatured peroxidase, enzyme refolding was accelerated in PEF and 60%
of initial activity was observed after 12 kV/cm and 30 s of PEF
treatment although spontaneous refolding of this enzyme resulted in
40% of initial activity. On the other hand, when PEF was applied to
thermal denatured lactate dehydrogenase (LDH), further PEF-induced
inactivation was observed. It was suggested that the influence of PEF
is dependent on the type of enzyme.
In halophiles, protein stability and function are maintained by
increased ion binding and glutamic acid content, both allowing the
protein inventory to compete for water at high salt. Acidophiles and
alkalophiles show neutral intracellular pH; proteins facing the
outside extremes of pH possess anomalously high contents in ionizable
These facts suggest that globular proteins should be maximally stable
around room temperature. Twenty-six of these are unique, and 20 of the
26 are maximally stable around room temperature irrespective of their
structural properties, the melting temperature, or the living
temperatures of their source organisms. Their average temperature of
maximal stability is 293 ± 8 K (20 ± 8 °C). The average energetic
contribution of the individual amino acids toward protein stability
decreases with an increase in protein size.
Analysed in terms of their effect on the protein structure, the ways
in which thermophilic organisms obtain relative stabilization of their
proteins can be classified as follows:
- increase of compactness and better packing
- increase of electrostatic interactions
- additional hydrogen bonds
- additional disulphide bridges
- increasing hydrophobic interactions
- protein microenvironment
The rational modification of protein stability is an important goal of
protein design. Protein surface electrostatic interactions are not
evolutionarily optimized for stability and are an attractive target
for the rational redesign of proteins. We show that surface charge
mutants can exert stabilizing effects in distinct and unanticipated
ways, including ones that are not predicted by existing methods, even
when only solvent-exposed sites are targeted. Individual mutation of
three solvent-exposed lysines in the villin headpiece subdomain
significantly stabilizes the protein, but the mechanism of
stabilization is very different in each case. One mutation
destabilizes native-state electrostatic interactions but has a larger
destabilizing effect on the denatured state, a second removes the
desolvation penalty paid by the charged residue, whereas the third
introduces unanticipated native-state interactions but does not alter
electrostatics. Our results show that even seemingly intuitive
mutations can exert their effects through unforeseen and complex
These results suggest that surface charge-charge interactions are
important for protein stability and that rational optimization of
charge-charge interactions on the protein surface can be a viable
strategy for enhancing protein stability.
We have discovered a novel property of single-walled carbon nanotubes
(SWNTs)their ability to stabilize proteins at elevated temperatures
and in organic solvents to a greater extent than conventional flat
supports. Experimental results and theoretical analysis reveal that
the stabilization results from the curvature of SWNTs, which
suppresses unfavorable protein-protein lateral interactions. Our
results also indicate that the phenomenon is not unique to SWNTs but
could be extended to other nanomaterials. The protein-nanotube
conjugates represent a new generation of active and stable catalytic
materials with potential use in biosensors, diagnostics, and bioactive
films and other hybrid materials that integrate biotic and abiotic
The main chain to side chain salt bridge between the N-terminus and
Glu 14 was, however, found to stabilize PFRD-XC4 by 1.5 kcal mol-1.
The entropic cost of making a surface salt bridge involving the
protein's backbone is reduced, since the backbone has already been
immobilized upon protein folding.