I was wondering how protein denaturation works.

  1. Are there covalent bonds, such as disulfide bridges involved, or is it based purely on non-covalent bonds such as hydrogen bonds? Why is denaturation irreversible in most cases if only non-covalent bonds are involved?
  2. Is it possible to denature protein by rapid changes in electromagnetic field or pressure? (The articles I have read so far mention only stress factors like sudden pH, osmolarity, temperature changes...)
  3. How can I protect a protein against denaturation? e.g. in PCR we use a heat resistant DNA polymerase, so certain amino acid sequences might protect against heat denaturation, but I need reassurance about this.

3 Answers 3


Really the question how does protein folding work? But let me answer your questions...

1) Very few proteins have disulfide bonds (usually secreted proteins) or really any covalent bond stabilizing the amino acid chain beyond the bonds that make up the polypeptide itself. Denaturation is only reversible in relatively few cases in fact. A few proteins, usually very small ones can be nursed back into a native folded state from an unfolded one, and then only a percentage of the sample will reattain the folded state.

2) Sure. In fact pressure changes the hydrogen bonded structure of the water and also therefore the thermodynamics of protein structure and has been used to study protein folding. Generally the electomagnetic field does not affect the state of the protein fold. I cite the fact that many many proteins have been studied via nuclear magnetic resonance, in which the proteins have been inserted into some of the most powerful magnets that can fit into a reasonably sized room. That is not to say that the protein function might not be affected by such a field. I'm sure by the time the field is large enough to ionize water or the peptide change you would see something... so you can always push things too the breaking point.

3) Many proteins from thermophilic organisms are more resistant to denaturation and companies actually engineer proteins to be resistant to all sorts of outrageous conditions and still be folded and functional. Laundry detergent is full of crystallized enzymes that will sit happily in detergent for a long time. I don't even know if these products have a shelf life.

Overall proteins are vulnerable to denaturation for a good reason- the cell degrades them when it doesn't need their function and can recycle them for their component amino acids. If they were all this robust the cell would starve to death quickly. If there is a biological role to a protein that is denatured and then refolds itself it is only for extremely rare situations. Proteins for the most part don't fall apart once they are folded and if they do, they are done.

Some possible exceptions: some antibiotic peptides which fold into pores that kill their targets and the amyloid plaque which takes on a different fold when in the brain which is associated with - but may not cause - Alzheimer's.

  • 1
    $\begingroup$ Some RNAse enzymes are capable of renaturing, makes it annoying to get rid of them. $\endgroup$
    – user137
    Commented Oct 17, 2014 at 19:11
  • 1
    $\begingroup$ "Very few proteins have disulfide bonds or really any covalent bond stabilizing the amino acid chain " - can you add some reference about this? e.g. insuline, egg yolk proteins, gluten, hair proteins contain disulfide bonds. I guess every protein with multiple cys can and maybe does have that, so I don't think it is rare. This part of question was about: do primary bonds form by denaturation or not? If not, then why is it irreversible? I don't think you can explain that only with secondary structural changes. e.g. by egg yolk it becomes white, so I guess there is protein aggregation involved. $\endgroup$
    – inf3rno
    Commented Oct 17, 2014 at 21:17
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    $\begingroup$ "Generally the electomagnetic field does not affect the state of the protein fold. " I hardly believe that, afaik based on the pH amino acids can have negative or positive charges, so the EM field can pull one end of the protein chain and repel the other end, but I think most of the proteins are not affected, because there is an even distribution of +/- charged amino acids along the chain. But that's just my theory. I can accept this part of your answer. $\endgroup$
    – inf3rno
    Commented Oct 17, 2014 at 21:22
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    $\begingroup$ "companies actually engineer proteins to be resistant to all sorts of outrageous conditions" - That part of the question was about how to engineer such proteins in theory. I suspected, that the technology already exists. $\endgroup$
    – inf3rno
    Commented Oct 17, 2014 at 21:29
  • $\begingroup$ added references. i misunderstood what you meant by electromagnetic field. the specific arrangement of charges can be quite important to a protein... usually this is seen as direct couloumbic repulsion or complementarity. most of the long range electrical fields from protein charges affect how proteins interact with each other. a harder question to answer definitively. $\endgroup$
    – shigeta
    Commented Oct 18, 2014 at 4:39

Are you taking in an in vitro context for preventing protein denaturation after protein isolation from for example E. coli or are you more worried about proteins in the context of the whole cell?

I'm no expert in the protein folding/conformation studies but from laboratory based prospective if you want to achieve denaturation for experimental purposes, you treat your samples with SDS and high heat (~ 100 oC for 10 min) to eliminate H-bonds and to get rid of disulphide bonds, you use a reducing agent such as beta-ME or DTT, which is commonly found in molecules such as Ab or cell surface receptors such as EGFR so obviously for western-blot experiments which you need your disulphide bridges to be preserved you do not use reducing agents but you still heat your samples and treat with SDS to get rid of the hydrogen bonds and linearalise your protein.

Based on this study, which used BSA and β-lactoglobulin, denaturation caused by high pressure is similar to that caused by the cleavage of hydrogen bonds with urea or guanidine hydrochloride so, yes, rapid changes in environmental conditions can have denaturing effects similar to chemical based agents.

If you are trying to prevent protein denaturation in a whole cell, then you need to treat them with cryo-preservation media containing usually 10% DMSO such as this. If you are working just with proteins alone, the best method is to work with your samples freshly prepared (lysed etc) and when isolating your proteins for future used, snap freeze them in liquid nitrogen and store at -80 oC. If your proteins are attached to beads such as GST-tagged proteins, then store your beads in a buffer congaing glycerol and put at -20 oC. I use 50% glycerol (v/v) and it works well for me! If however this response does not answer your particular question or concern, please edit and elaborate on what exactly you are worried about in your line of work and I shall modify my response accordingly. Hope this helped in some way!

  • $\begingroup$ Thanks you answer it was interesting. +1 I am not worried about anything, I just want to learn more about the details of how denaturation works. I want to refresh my memory about bioengineering, I skipped the last 3 years, since I learned software engineering. Instead of relearning the whole stuff I thought it would be better to ask more detailed questions about one or more topics... I meant in vitro proteins, for example egg white, etc..., and it was a theoretical question, but it was good to see some laboratory practices again. Just be careful with mercaptoethanol, it can cause cancer! :-) $\endgroup$
    – inf3rno
    Commented Oct 17, 2014 at 21:08

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 conformations.

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 core cluster

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 unaffected.

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 thermodynamical conditions.

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 entropically driven.

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 protein dynamics.

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 dairy products.

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 ultrasound treatment.

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 amino acids.

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
  • glycosylation

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 interactions.

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 components.

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.

  • $\begingroup$ This is an impressive list of references, but how many of these are available to the general public outside an university? $\endgroup$
    – Chris
    Commented Nov 7, 2014 at 8:36
  • $\begingroup$ @Chris I think about 5-10% (bigger libraries usually have access to scientific articles as well). I too don't have access to the full text, just the abstracts in most of the articles, but it is usually enough to add one or more important sentences to my post. It is pretty hard to collect the information this way (as you can see I used around 30 references for a single page). Reading books is not enough either if you want to be up to date... :S $\endgroup$
    – inf3rno
    Commented Nov 7, 2014 at 14:37
  • $\begingroup$ Longest Biology.SE answer! Good job! $\endgroup$
    – TanMath
    Commented Oct 16, 2015 at 23:59
  • $\begingroup$ @TanMath Actually 5/10 is mine answer from your list. Does not matter. I just like using long quotes and lots of references. $\endgroup$
    – inf3rno
    Commented Oct 17, 2015 at 7:26

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