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How does protein denaturation work?

How does protein denaturation work?


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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.

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.


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!


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

References:

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…

  • 1996 - The Source of Stability in Proteins
  • 2009 - Charge-charge interactions in the denatured state influence the folding kinetics of ribonuclease Sa
  • 2006 - Why Are Proteins Charged? Networks of Charge-Charge Interactions in Proteins Measured by Charge Ladders and Capillary Electrophoresis

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.

  • 1996 - Forces contributing to the conformational stability of proteins.
  • 2005 - Do all backbone polar groups in proteins form hydrogen bonds?
  • 2011 - Backbone-Driven Collapse in Unfolded Protein Chains
  • 2006 - Hydrophobic collapse in (in silico) protein folding
  • 2002 - Thermodynamic Consequences of Burial of Polar and Non-polar Amino Acid Residues in the Protein Interior
  • 2003 - Thermodynamics of Heat Activation of Single Capsaicin Ion Channels VR1
  • 2003 - Temperature Range of Thermodynamic Stability for the Native State of Reversible Two-State Proteins
  • 2009 - Protein Cold Denaturation as Seen From the Solvent
  • 2010 - Construction of Peptoids with All Trans-Amide Backbones and Peptoid Reverse Turns via the Tactical Incorporation of N-Aryl Side Chains Capable of Hydrogen Bonding
  • 2002 - Energy of Hydrogen Bonds Probed by the Adhesion of Functionalized Lipid Layers
  • 2012 - How, when and why proteins collapse: the relation to folding
  • 2011 - Inversion of the Balance between Hydrophobic and Hydrogen Bonding Interactions in Protein Folding and Aggregation
  • 2011 - Membrane protein folding: how important are hydrogen bonds?

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.

  • 2013 - Molecular dynamics simulation indicating cold denaturation of beta-hairpins.
  • 2012 - Cold-Induced Changes in the Protein Ubiquitin
  • 2008 - Microscopic Mechanism for Cold Denaturation
  • 2009 - Hydrophobicity at low temperatures and cold denaturation of a protein
  • 2012 Key stabilizing elements of protein structure identified through pressure and temperature perturbation of its hydrogen bond network
  • 2012 - Unifying Microscopic Mechanism for Pressure and Cold Denaturations of Proteins
  • 2012 - Role of Hydrophobic Hydration in Protein Stability: A 3D Water-Explicit Protein Model Exhibiting Cold and Heat Denaturation
  • 2011 - Molecular mechanisms of the anomalous thermal aggregation of green fluorescent protein
  • 2011 - Analysis of the Mechanism of Lysozyme Pressure Denaturation from Raman Spectroscopy Investigations, and Comparison with Thermal Denaturation
  • 2008 - Cold- and Pressure-Induced Dissociation of Protein Aggregates and Amyloid Fibrils
  • 2009 - The Behavior of the Hydrophobic Effect under Pressure and Protein Denaturation
  • 2004 - Reversible Temperature and Pressure Denaturation of a Protein Fragment: A Replica Exchange Molecular Dynamics Simulation Study

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.

  • 2008 - Structure and Energetics of the Hydrogen-Bonded Backbone in Protein Folding
  • 2008 - Urea, but not guanidinium, destabilizes proteins by forming hydrogen bonds to the peptide group
  • 2011 - Backbone and Side-Chain Contributions in Protein Denaturation by Urea
  • 2009 - On the mechanism of SDS-induced protein denaturation
  • 1995 - Hydrogen Bonds and the pH dependence of Ovomucoid Third Domain Stability
  • 2004 - Effects of Chaotropic and Kosmotropic Cosolvents on the Pressure-Induced Unfolding and Denaturation of Proteins:? An FT-IR Study on Staphylococcal Nuclease
  • 2002 - The hydration structure of guanidinium and thiocyanate ions: Implications for protein stability in aqueous solution

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.

  • 2011 - Effects of ultrasound on the thermal and structural characteristics of proteins in reconstituted whey protein concentrate
  • 2011 - Effects of low frequency ultrasound on some properties of fibrinogen and its plasminolysis

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.

  • 2014 - Real-time assessment of possible electromagnetic-field-induced changes in protein conformation and thermal stability
  • 2007 - Effects of pulsed electric fields on physicochemical properties of soybean protein isolates
  • 1997 - Effects of high field electric pulses on the activity of selected enzymes
  • 2007 - Influence of pulsed electric field on various enzyme activities

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.

  • 2000 - Stability and stabilization of globular proteins in solution
  • 2002 - Maximal Stabilities of Reversible Two-State Proteins
  • 2014 - Enzyme Thermostabilization: the State of the Art
  • 2012 - Rational modification of protein stability by targeting surface sites leads to complicated results
  • 2012 - Disulfide Bonding in Protein Biophysics
  • 2011 - Methanol Strengthens Hydrogen Bonds and Weakens Hydrophobic Interactions in Proteins - A Combined Molecular Dynamics and NMR study
  • 2011 - Contribution of Hydrophobic Interactions to Protein Stability
  • 1997 - Protein thermal stability, hydrogen bonds, and ion pairs
  • 2005 - Thermal stability of proteins.
  • 2004 - Protein structure, stability and solubility in water and other solvents.
  • 2000 - Rational Modification of Protein Stability by the Mutation of Charged Surface Residues
  • 2006 - Protein Stability and Surface Electrostatics:? A Charged Relationship
  • 2003 - Contribution of Surface Salt Bridges to Protein Stability: Guidelines for Protein Engineering
  • 2000 - Influence of Sucrose on the Thermal Denaturation, Gelation, and Emulsion Stabilization of Whey Proteins
  • 2006 - Increasing Protein Stability through Control of the Nanoscale Environment
  • 2000 - Contribution of Surface Salt Bridges to Protein Stability
  • 2003 - Elastic coupling of integral membrane protein stability to lipid bilayer forces

Protein Denaturation in Foam

The aim of this study was to elucidate the mechanism by which protein molecules become denatured in foam. It was found that damage to the protein is mainly due to surface denaturation at the gas-liquid interface. A fraction of the molecules adsorbed do not refold to their native state when they desorb. The degree of denaturation was found to correlate directly with the interfacial exposure, which, for mobile or partially mobile interfaces, is increased by drainage. Experiments with two different proteins showed that, under the conditions of the tests, around 10% of BSA molecules which had adsorbed at the surface remained denatured when they desorbed. For pepsin the figure was around 75%. Oxidation, which was previously thought to be a major cause of protein damage in foam, was found to be minimal. Neither do the high shear stresses in the liquid bulk encountered during bubble bursting cause denaturation, because energy is dissipated at a much greater length scale than that of the protein molecule. Copyright 1999 Academic Press.


Denaturation of Proteins (with Denaturing Agents)

Denaturation may be defined as the disruption of the secondary, tertiary and quarternary structure of the native protein resulting in the alterations of the physical, chemical and biological characteristics of the protein by a variety of agents.

The native proteins are said to be the proteins occurring in animal and plant tissues. They pos­sess many characteristic properties such as solubil­ity, viscosity, optical rotation, sedimentation rate, electrophoretic mobility etc. For an oligomeric protein, denaturation may involve dissociation of the protomers with or with­out subsequent unfolding or with or without un­dergoing changes in protomer conformation.

Denaturing Agents:

Heat, surface action, ul­traviolet light, ultrasound, high pressure etc.

Acids, alkalis, heavy metal salts, urea, ethanol, guanidine de­tergents etc. Urea and guanidine probably interfere with the hydrogen bonds between peptide linkages. Acids and alkalis probably attack directly the hy­drogen bonds in the secondary and tertiary struc­ture of proteins.

Physical Alterations:

Many proteins, especially of the globular type, can be crystallized in the native state. But dena­tured proteins cannot be crystallized.

Chemical Alterations:

The denatured protein is greatly decreased in solubility at its isoelectric point. The chemical groups are exposed to chemical reactions and more readily detected as a result of the unfolding proc­ess in denaturation. Among these are sulphydryl group of cysteine, the disulphide group of cystine and the phenolic group of tyrosine.

Biological Alterations:

The digestibility of certain denatured proteins by proteolytic enzymes is increased. Enzymatic or hormonal activity is usually destroyed by dena­turation. The antigenic or antibody functions of proteins are frequently altered.

If the denaturation is severe, the protein mol­ecules become insoluble and precipitation results as well as the changes in the properties of the pro­teins are permanent and “irreversible”. In case of mild denaturation, there is “reversible denatura­tion” leading to the slight changes in the proper­ties of the protein which can be restored to the na­tive state after suitable treatment.

1. The precipitation of the native protein as a result of denaturation is used to advan­tage in the clinical laboratory.

2. Blood or serum samples to be analysed for small molecules (e.g., glucose, uric acid, drugs) generally are first treated with ac­ids such as trichloroacetic acid, phosphotungstic acid or phosphomolybdic acid to precipitate most of the proteins present in the sample. This is removed by cen­trifugation and the protein-free supernatant liquid is then analysed.

3. Denaturation is used to know the enzyme catalysed reaction of an extract at the loss of the enzyme activity when boiled or acidified.

Denaturation and Renaturation of Proteins:

Bovine ribonuclease of single polypeptide chain of 124 amino acid residues with small molecular weight contains four disulphide bonds. When it is treated with β-mercaptoethanol in 8 M. urea, the disulphide bonds are reduced to -SH groups as a result of the denaturation of the en­zyme and the enzyme activity is also lost.

Denatured ribonuclease, when freed from urea and β-mercaptoethanol by dialysis, slowly regains enzymic activity as the SH groups are oxidized by oxygen of air to form S-S bonds. But if the reduced ribonuclease in 8 M. urea solution is re-oxidized it loses its enzymic activity almost completely as wrong disulphide bonds are formed resulting in ‘scrumbled’ ribonuclease.

Similarly, when egg albumin is heated till it is coagulated, the denaturation is irreversible and the secondary and tertiary structure of the proteins are completely lost resulting in a mixture of ran­domly arranged polypeptide chains.


Odorant Binding and Chemosensory Proteins

Nadja Hellmann , in Methods in Enzymology , 2020

3.1 Equilibrium conditions and the issue of reversibility

Strictly speaking, when investigating protein denaturation , thermodynamic equilibrium means that the fractions of protein in the various states (native, intermediate, denatured) are only determined by the conditions (e.g., denaturant concentration, temperature) and not by the path which led to this state (e.g., per unfolding or per refolding). Thus, unfolding and refolding curves should not show any hysteresis, but should overlap. Then the process is reversible, and both curves are in equilibrium.

To achieve equilibrium conditions, as indicated by a temporally stable spectroscopic signal, it might take a while: for chemical denaturation of OBPs often incubation times of 24 h were employed. The refolding process might take even longer and often displays considerable hysteresis ( Stepanenko et al., 2014, 2016b ). Typically, the refolding curve is the one which does not represent equilibrium conditions.

In thermal denaturation studies, different results might be obtained for different heating rates. If denaturation is slower than the change in temperature, the denatured fraction lags behind and the curves obtained do not represent equilibrium conditions.

Irreversible aggregation of the denatured protein tends to occur in thermal denaturation ( Parisi et al., 2005 ). If the temperature at which aggregation starts (Tagg) is considerably higher than the denaturation temperature (Tm), it is possible to separate the two processes, and restrict the analysis to the range below Tagg. Not unfrequently, however, both processes take place within a narrow temperature range, and the obtained value of Tm might be distorted.


How Does Denaturation Affect the Function of a Protein?

Denaturation causes a protein to lose its biological function. For example, a denatured enzyme would no longer be able to catalyze a reaction.

Denaturation does not alter the protein's structure, nor does it hydrolyze the peptide bonds. While it causes the protein's structure to unfold, the amino acids forming it remain. Following denaturation, a protein cannot fulfill its biological role. This means an enzyme can no longer catalyze its target reaction, and insulin cannot target molecules to aid the movement of glucose into cells. When using heat to denature a protein, there are some instances where cooling it down can restore its function. However, in most cases the alteration is permanent.

There are several ways to denature a protein. Using salt, urea, acids and bases and heat interrupts the bonds between hydrogen and amides, which in turn causes it to lose its structure. When it comes to tertiary proteins, this may mean losing a hydrogen bond, disulfide bond, salt bridge and non-polar covalent bonds. Because of this, there is a broad number of substances that lead to denaturation. Heat can be used to break non-polar covalent bonds and hydrogen bonds. It is because of this that heat is a useful tool for sterilizing medical supplies and food preparation areas.


Protein denaturation

When a solution of a protein is boiled, the protein frequently becomes insoluble—i.e., it is denatured—and remains insoluble even when the solution is cooled. The denaturation of the proteins of egg white by heat—as when boiling an egg—is an example of irreversible denaturation. The denatured protein has the same primary structure as the original, or native, protein. The weak forces between charged groups and the weaker forces of mutual attraction of nonpolar groups are disrupted at elevated temperatures, however as a result, the tertiary structure of the protein is lost. In some instances the original structure of the protein can be regenerated the process is called renaturation.

Denaturation can be brought about in various ways. Proteins are denatured by treatment with alkaline or acid, oxidizing or reducing agents, and certain organic solvents. Interesting among denaturing agents are those that affect the secondary and tertiary structure without affecting the primary structure. The agents most frequently used for this purpose are urea and guanidinium chloride. These molecules, because of their high affinity for peptide bonds, break the hydrogen bonds and the salt bridges between positive and negative side chains, thereby abolishing the tertiary structure of the peptide chain. When denaturing agents are removed from a protein solution, the native protein re-forms in many cases. Denaturation can also be accomplished by reduction of the disulfide bonds of cystine—i.e., conversion of the disulfide bond (―S―S―) to two sulfhydryl groups (―SH). This, of course, results in the formation of two cysteines. Reoxidation of the cysteines by exposure to air sometimes regenerates the native protein. In other cases, however, the wrong cysteines become bound to each other, resulting in a different protein. Finally, denaturation can also be accomplished by exposing proteins to organic solvents such as ethanol or acetone. It is believed that the organic solvents interfere with the mutual attraction of nonpolar groups.

Some of the smaller proteins, however, are extremely stable, even against heat for example, solutions of ribonuclease can be exposed for short periods of time to temperatures of 90 °C (194 °F) without undergoing significant denaturation. Denaturation does not involve identical changes in protein molecules. A common property of denatured proteins, however, is the loss of biological activity—e.g., the ability to act as enzymes or hormones.

Although denaturation had long been considered an all-or-none reaction, it is now thought that many intermediary states exist between native and denatured protein. In some instances, however, the breaking of a key bond could be followed by the complete breakdown of the conformation of the native protein.

Although many native proteins are resistant to the action of the enzyme trypsin, which breaks down proteins during digestion, they are hydrolyzed by the same enzyme after denaturation. The peptide bonds that can be split by trypsin are inaccessible in the native proteins but become accessible during denaturation. Similarly, denatured proteins give more intense colour reactions for tyrosine, histidine, and arginine than do the same proteins in the native state. The increased accessibility of reactive groups of denatured proteins is attributed to an unfolding of the peptide chains.

If denaturation can be brought about easily and if renaturation is difficult, how is the native conformation of globular proteins maintained in living organisms, in which they are produced stepwise, by incorporation of one amino acid at a time? Experiments on the biosynthesis of proteins from amino acids containing radioactive carbon or heavy hydrogen reveal that the protein molecule grows stepwise from the N terminus to the C terminus in each step a single amino acid residue is incorporated. As soon as the growing peptide chain contains six or seven amino acid residues, the side chains interact with each other and thus cause deviations from the straight or β-chain configuration. Depending on the nature of the side chains, this may result in the formation of an α-helix or of loops closed by hydrogen bonds or disulfide bridges. The final conformation is probably frozen when the peptide chain attains a length of 50 or more amino acid residues.


Online Literature:

  1. K. A. Dill and J. L. MacCallum, The protein folding problem, 50 years on, Science 338, 1042-1046 (2012). (PDF) (Full Text Online) (podcast)
  2. Southall, N.T., K.A. Dill, and A.D.J. Haymet. A View of the Hydrophobic Effect. Journal of Physical Chemistry B 106: 521-533 (2002). (PDF)
  3. Summary of studies from small molecules (N-methyacetamide and benzene)

It is clear that proteins are not all that stable, and many contributions of varying magnitudes must sum to give the proteins marginal stability under physiological conditions. Hydrophobic interaction, defined in the new sense, must play a major role in stability. Also, since proteins are so highly packed compared to a lose denatured state, London Forces must also play a significant part. (Remember dispersion forces are short range and become most significant under conditions of closest packing.) Opposing folding is the chain conformational entropy just described. Since proteins are so marginally stable, even one unpaired buried ionic side chain, or 1-2 unpaired buried H bond donors and acceptors in the protein may be enough to "unravel" the native structure, leading to the denatured state.


Odorant Binding and Chemosensory Proteins

Nadja Hellmann , in Methods in Enzymology , 2020

3.1 Equilibrium conditions and the issue of reversibility

Strictly speaking, when investigating protein denaturation , thermodynamic equilibrium means that the fractions of protein in the various states (native, intermediate, denatured) are only determined by the conditions (e.g., denaturant concentration, temperature) and not by the path which led to this state (e.g., per unfolding or per refolding). Thus, unfolding and refolding curves should not show any hysteresis, but should overlap. Then the process is reversible, and both curves are in equilibrium.

To achieve equilibrium conditions, as indicated by a temporally stable spectroscopic signal, it might take a while: for chemical denaturation of OBPs often incubation times of 24 h were employed. The refolding process might take even longer and often displays considerable hysteresis ( Stepanenko et al., 2014, 2016b ). Typically, the refolding curve is the one which does not represent equilibrium conditions.

In thermal denaturation studies, different results might be obtained for different heating rates. If denaturation is slower than the change in temperature, the denatured fraction lags behind and the curves obtained do not represent equilibrium conditions.

Irreversible aggregation of the denatured protein tends to occur in thermal denaturation ( Parisi et al., 2005 ). If the temperature at which aggregation starts (Tagg) is considerably higher than the denaturation temperature (Tm), it is possible to separate the two processes, and restrict the analysis to the range below Tagg. Not unfrequently, however, both processes take place within a narrow temperature range, and the obtained value of Tm might be distorted.


Protein

Proteins are formed of amino acid residues (more than 100 amino acids) linked together by peptide bonds, chemically, polymerization of amino acids into protein is a dehydration reaction, they are of high molecular weight (more than 5000) colloidal in nature, non-dialysable, and heat-labile. Each protein has a unique, precisely defined amino acids sequence, amino acid sequences are important for several reasons:

  • Knowledge of the amino acids sequence of proteins helps in clearing its mechanism of action (e.g. The catalytic mechanism of enzyme).
  • Alteration in amino acids sequence can produce abnormal function and disease e.g. Sickle cell disease.

Protein structure

Bonds responsible for protein structure are:

I. Covalent bonds

  1. Peptide bonds (Amide bonds):The carboxylic group of one amino acid combines with the amino group of another amino acid (with the removal of a molecule of water). This is a rigid bond, strong, no rotation of protein molecule can occur around this bond (that connects C and N atoms), so it stabilizes the protein structure, this bond occurs in transform, and all the 4 atoms lie in the same plane (i.e. are coplanar).Peptide bonds are not broken in the denaturation of proteins i..e. on expoure to heat or x-ray or on shaking, they can be broken by enzymatic action or by strong acid or base at elevated temperature.
  2. Disulfide bonds (-S-S-):They occur between 2 cysteine residues in the same polypeptide chain or in different polypeptide chains, it is a very stable bong resists conditions usual for protein denaturations.

II. Non-covalent bonds

These are weak bonds, can be separated easily, however, the large numbers of these bonds in the protein molecule add up to the forces favoring protein folding.

  1. Hydrogen bonds are formed when a sharing of hydrogen atom occurs between the hydrogen of -NH group and the carbonyl oxygen of different peptide bonds, hydrogen bonds may be formed between polar uncharged R groups e.g. -OH with each other or with water.
  2. Hydrophobic interactions:The non-polar side chains of neutral amino acids tend to be introduced to the inside of the protein molecule exposed to water, they are not true bonds but interactions that help to stabilize the protein structure.
  3. Electrostatic bonds (ionic interaction or salt bridge): These bonds occur between the charged group of side chains of amino acids, (NH3 + of basic amino acids and COO¯ of acidic amino acids)
    They are either:
    a. Repulsive: If the interactions between the side chains are of the same sign, [both are (+) or both are (-)].
    b. Attractive: If the interactions occur between side chains of different charges [i.e. one is (+) and the other is (-)].
The conformation of proteins (Orders of protein structure)

In its native form, protein molecule has a characteristic three-dimensional shape (primary, secondary, tertiary structure), which is required for its specific biological function or activity, proteins formed of two or more polypeptide chains have a quaternary structure.

Protein structure

1- Primary structure of proteins

This refers to the number and sequence of amino acids in the polypeptide chain or chains linked by peptide bonds, understanding of primary structures of proteins is important because many genetic diseases result with abnormal amino acid sequences. The amino acids sequences are read from N-terminal (amino acid number 1) to C-terminal ends of the peptide, the primary structure of proteins determines the secondary and tertiary structures which are essential for protein functions.

2- Secondary structure of proteins

Coiling, folding, or bending of the polypeptide chain leading to specific structure kept by interactions of amino acids close to each other in the sequence of the polypeptide chain, there are two main regular forms of secondary structure: α-helix and β-pleated sheets, other forms may be found.

3- Tertiary structure of proteins

It is the three-dimensional structure of each polypeptide chain, there are two main forms of tertiary structure: fibrous and globular types.

Domains are the functional and three-dimensional structural units of a polypeptide, folding of the peptide chain within a domain is independent of folding in other domains, thus each domain has the characteristics of a small compact globular protein, polypeptides that are greater than 200 amino acids generally consist of two or more domains,

The domains are usually connected with relatively flexible areas of protein. Interactions stabilizing tertiary structure include disulfide bonds, hydrophobic interactions, hydrogen bonds, and ionic interactions.

4- Quaternary structure of proteins

Certain polypeptides will aggregate to form one functional protein, proteins possess quaternary structure if they consist of 2 or more polypeptide chains, structurally identical, or totally unrelated united by non-covalent bonds (hydrogen, electrostatic bonds, and hydrophobic interaction), such proteins are termed oligomers, and the individual polypeptide chain is termed monomer or subunit, this protein will lose its function when the subunits are dissociated.
e.g. Hemoglobin is an example of protein present in the quaternary structure, it is a tetramer having 2α chains and 2β chains.

Denaturation of proteins

It is the loss of native structure (natural conformation) of protein by many physical or chemical agents leading to changes in the secondary, tertiary, and quaternary structure of proteins due to rupture of the non-covalent bonds (hydrogen bonds, hydrophobic bonds, and electrostatic bonds and may be disulphide, but not peptide bonds), with loss of biological activity. Denaturation disrupts all orders of protein structure except the primary structure.


What is the Role of SDS

The R-groups of amino acids in a particular protein may bear either positive or negative charge, making the protein an amphoteric molecule. Therefore, in the native state, different proteins with the same molecular weight migrate at different speeds on the gel. This makes the separation of proteins in the polyacrylamide gel difficult. The addition of SDS to the protein denatures the proteins and covers them in a uniformly-distributed, net negative charge. This allows the migration of proteins towards the positive electrode during electrophoresis. In other words, SDS linearizes the protein molecules and masks the various types of charges on R-groups. In conclusion, the charge to mass ratio in SDS-coated proteins is same hence, there will be no differential migration based on the charge of the native protein. A SDS-PAGE of red blood cell membrane proteins is shown in figure 3.

Figure 3: SDS-PAGE

In addition to SDS-PAGE, SDS is used as a detergent in nucleic acid extractions for the disruption of the cell membrane and dissociation of nucleic acid: protein complexes.

Conclusion

SDS is an anionic detergent used as a detergent in various types of biotechnological techniques. It denatures the tertiary structure of a protein to produce a linear protein molecule. Furthermore, it binds to the denatured protein in a uniform manner, providing a uniform charge to mass ratio to all types of proteins. A net negative charge is given to the protein molecule by SDS by masking the charges on R-groups of amino acids of the protein. Hence, SDS allows the separation of proteins based on their molecular weight on a PAGE as the charge is proportional to the molecular weight of the denatured proteins by SDS.

Reference:

1. “How SDS-PAGE Works.” Bitesize Bio, 16 Feb. 2018, Available here.

Image Courtesy:

1. “SDS with structure description” By CindyLi2016 – Own work (CC BY-SA 4.0) via Commons Wikimedia
2. “Protein-SDS interaction” By Fdardel – Own work (CC BY-SA 4.0) via Commons Wikimedia
3. “RBC Membrane Proteins SDS-PAGE gel” By Ernst Hempelmann – Ernst Hempelmann (Public Domain) via Commons Wikimedia

About the Author: Lakna

Lakna, a graduate in Molecular Biology & Biochemistry, is a Molecular Biologist and has a broad and keen interest in the discovery of nature related things


Watch the video: Biochemistry self experiment video - protein denaturation (May 2022).