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Protein Denaturation and Renaturation



Protein Denaturation or Unfolding

Native proteins are only marginally stable under physiological conditions

The free energy required to denature them is ~0.4 kJ/mol of amino acid residues

Low conformational stabilities of native proteins make them easily susceptible to denature by altering balance of weak non-bonding force that maintain the native conformation Exposing soluble or globular proteins to extremes of pH or to high temperatures for only short periods causes most of them to undergo a physical change called denaturation, in which the most visible effect is a decrease in solubility

Denaturation is a physical change, which involves unfolding of the characteristics native folded structure of polypeptide chain of globular protein molecules

Causes disorganization of internal structure of protein

Involves modification in secondary, tertiary or quaternary structure

Random coil conformation is achieved

Primary structure remains intact

Driving force increase in entropy

Early unlocking of tertiary structure deletes a large number of bonds holding the structure together but increases the randomness only insignificantly later stages of denaturation leads to larger increase in entropy



Approaches used to define the concept of denaturation can be classified into two types

Molecular actual structural changes taking place

Operational changes in measurable properties

Properties of denatured protein

Changes in properties of protein upon denaturation

Structure differs from native state

Modification in secondary, tertiary or quaternary structure

Changes in secondary bonds such as ion dipole, hydrogen, VDW, hydrophobic, in rotational positions about single bonds which are controlled by secondary bond structures

Primary structure remains intact

No covalent bonds in the backbone are broken

No peptide bonds are broken

Change in size and shape

No change in molecular weight

Decrease in solubility

Denatured protein loses its characteristic biological activity

Most protein molecules retain their biological activity only within a very limited range of

temperature or pH

Cessation of biochemical activity as enzymes or hormones

Increased activity of some radicals present in molecule as SH group of cysteine, -S-S- bond of cystine, phenolic group of tyrosine

New ionizable R groups become available

Change in optical rotation in the direction of increased levorotation

Alteration of surface tension

Loss of antigenicity

Ease of hydrolysis

Increased activity of protease

Upon unfolding new sites are exposed to the action of certain proteolytic enzymes

Altered water binding capacity

Destruction of toxins

Increased viscosity due to aggregate formation

Inability to crystallize

Decreased stability

Increased flexibility random coil varying φ and ψ angles

Mostly followed by coagulation tends to form aggregates and precipitated out from


May be irreversible or reversible

Denaturation is cooperative

Partial denaturation is followed by complete denaturation

Any partial unfolding of structure destabilizes the remaining structure which collapses into random coil

An unfolded protein makes most of its H-bonds with the water molecules of aqueous

solvent (water is a strong H-bonding donor and acceptor)

Temperature at the midpoint of denaturation is Tm or melting temperature

For most proteins, Tm is well below 100°C

Factors affecting protein denaturation

Physical agents


Heat treatment

Cooling and freezing operations

Freeze thaw

Interface (liquid air or liquid liquid interface)

Mechanical action

Agitation (Shaking)

Stirring and mixing: shear forces


High pressure



High hydrostatic pressures

UV rays

Chemical agents

In chemical denaturation, the secondary bonds (ion dipole, H-bonds, hydrophobic

interactions, rotation of single bonds φ and ψ) holding the protein segments together are disrupted by some chemicals capable of forming equally strong or stronger bonds with the

groups holding the conformation together

Extremes of pH (& Acids or Bases)

Inorganic salts


Water miscible organic solvents (& Dielectric constant)

Reducing agents

Anionic detergents / Surface active agents

Organic solvents

Ionizing radiations

Aromatic anions


Factors affecting protein denaturation Temperature

Most globular proteins denature when heated above 60-70°C

As temperature is increased, a number of bonds are weakened

Increase in temperature

Long range interactions necessary for tertiary structure are weakened and broken

Protein obtains a more flexible structure

Groups are exposed to solvent If heating ceases at this stage, the protein should be able to readily refold to native structure

Continued heating

Some of the cooperative H-bonds that stabilize helical structure begin to break

[H-bonds are broken due to increased vibrational and translational energy]

Water can interact with and form new H-bonds with the amide N and carbonyl O of peptide bond

Water further weakens nearby H-bonds by causing an increase in the effective D near them

Helical structure is broken

Hydrophobic groups (nonpolar R groups) are exposed to solvent


Intermolecular clustering, Aggregation and Precipitation

[Protein to minimize its free energy by burying as many hydrophobic groups while exposing as many polar groups as possible to the solvent]

Temperatures at which various proteins denature vary enormously

Some proteins are denatured at elevated temperature, some at very low temperatures, some at

temperature few degrees higher than that at which they function, some at very high temperature

(e.g., gluten)

Heat denaturation is all-or-none phenomenon

Hyperthermophilic bacteria grow at 122°C has compact proteins and increased number of salt




Factors affecting protein denaturation Liquid-liquid & Liquid-air interface

When proteins are exposed to either liquid-air or liquid-liquid interface, denaturation can occur

At a liquid-liquid or liquid-air interface, the protein comes into contact with a hydrophobic environment

Protein exposed to liquid-liquid interface

Protein comes in contact with hydrophobic environment

Protein allowed to remain at this interface for a period of time

Protein tends to place as many of their hydrophobic groups as possible in non-aqueous layer and as

many of their charged groups in water layer

Protein unfolds An association of hydrophobic groups causes the protein to aggregate



If energy is applied to cause shear, the process will be accelerated

The shear can cause the protein to unfold

Thus exposing its hydrophobic groups to non-aqueous phase

It can also increase the interfacial area between two phases

It allows more proteins to come into contact with non-aqueous phase

This folding is essentially irreversible because of large energy barriers

Even if the phases should separate and the protein is forced into aqueous phase, the protein will not regain its original structure

The presence of this denatured protein will serve as a barrier to further denaturation

The same forces are in operation when the protein migrates to liquid-air interface

Hydrophobic groups tend to associate in air and the protein unfolds

The presence of shear causes to help unfold the protein and to introduce more air into solution

Factors affecting protein denaturation

Mechanical action [Agitation (Shaking); Stirring and mixing: shear forces;

Ultrasound; High pressure; Rubbing; Homogenization, Hydrostatic pressure], UV

Mechanical action

Deformation and exposure of hydrophobic residues aggregation

Hydrostatic pressure

Pressure exerted by a fluid at equilibrium due to force of gravity

5000-10000 atm

Destabilizes hydrophobic aggregates

UV light

Supplies kinetic energy causing their atoms to vibrate more rapidly and disrupting weak H-bonds and dispersion forces



Factors affecting protein denaturation pH (Acids and Bases)

Most proteins at physiological pH are above their isoelectric points and have a net negative charge

Acid or bases / pH change disrupts salt bridges held together by ionic charges

pH variation alters ionization states of R groups which changes protein charge distribution and H-bonding requirements

If pH = pI, net charge is zero

If pH <<< pI, protein will lose its negative charge and contain net positive charge

If pH >>> pI, protein will have net negative charge

At pH <<< pI (Low pH)

Proteins have net positive charge

pI ( Low pH ) Proteins have net positive charge ↓ Like charges repel each other

Like charges repel each other

Proteins do not aggregate

In areas of large charge density

The intramolecular repulsion may be great enough

to cause unfolding of protein

In some cases

unfolding may be extensive enough to expose hydrophobic groups This leads to irreversible aggregation

Until this occurs, such unfolding will be largely reversible


The effects of high pH

are analogous to those of

low pH

The protein obtain a net negative charge

which can cause

unfolding and even aggregation


Factors affecting protein denaturation Inorganic salts (note: urea and guanidinium chloride are separated as denaturants)

Some salts

Stabilize native structure of protein (raise its Tm) (NH 4 ) 2 SO 4 , KH 2 PO 4

Have little effect NaCl, KCl

Destabilize protein KSCN, LiBr

Order of effectiveness of various ions in stabilizing a protein parallels with the capacity to salt-out protein (Hoffmeister series )


F - ~ SO 4 2- > H 2 PO 4 - > CH 3 COO - > Cl - > NO 3 - > Br - > ClO 4 - > I - > ClO 4 - > SCN - Cations

NH 4 + , Cs + > K + > Na + > Li + > Mg 2+ > Ca 2+ > Ba 2+ > guanidinium

Early members Kosmotropes

Late members Chaotropes

Effect of various ions is cumulative

GuSO 4 > GuCl > GuSCN

GuSO 4 stabilizes protein

GuCl is less potent denaturant than GuSCN

GuSCN is strong denaturant



Early members of series Kosmotropes

Stabilize proteins

Strengthens hydrophobic forces and hence increase the tendency of water to expel proteins

Acid precipitation (salting-out)

Example, NH 4 + , K + , SO 4 2-

Late members of series


Tend to denature protein

Tend to increase solubility in water

These increase the solubility of nonpolar substances in water

Disrupts hydrophobic interaction Their effectiveness as denaturing agents stems from their ability to disrupt hydrophobic interaction

Example, I - , ClO 4 - , SCN - , Li + , Mg 2+ , Ca 2+ , Ba 2+

Factors affecting protein denaturation Denaturants

Urea, guanidinium chloride denature protein by forming H-bonds to the protein groups that are stronger than H-bonds between the groups

Protein devoid of crosslinks treated with 8M urea or 6M guanidinium chloride

1. Single polypeptide protein

Complete unfolding

Assumes a random coil conformation

2. Multisubunit protein

Separation of subunits

3. Sulphydryl groups made accessible by unfolding of polypeptide(s)

Protein aggregation due to formation of S-S bridges between different proteins

[Such reactions may be inhibited by iodoacetate]

[Under these conditions, denatured molecules remain in solution and may revert into native molecules



if denaturing agent is slowly dialyzed away]

SRV 29

Factors affecting protein denaturation Water miscible organic solvents & Dielectric constant (D)

Solvent precipitation

Alcohol / acetone (less polar than water; miscible with water; neutral organic solvent) (has lower D than water)

Weakening of hydrophobic bonds of proteins (disrupt hydrophobic interaction) Own hydrophobic interaction with water (along with the effect of lower D)

Structure of protein changed


Lower D

Increases strength of all electrostatic interactions between molecules that were in contact with water






hydroxyl groups) poor

denaturants because their H-bonding ability

renders them

disruptive of water structure



Factors affecting protein denaturation

Reducing agents,

Detergents (surface active agents), Organic solvents

(formamide, dimethyl formamide, dichloroacetic acid DCA,

trichloroacetic acid TCA and their salts), Ionizing radiations

Reducing agents

Examples, β-mercaptoethanol and Dithiothreitol (DTT)

These disrupt S-S bonds and form thiol

Can be used in conjunction with urea or SDS to fully solubilize protein

Detergents (surface active agents)

Example, SDS

Disrupts H-bonding and hydrophobic interactions

Solvate denatured protein

Hydrophobically associate with nonpolar amino acids of proteins, thereby interfering with hydrophobic interactions responsible for protein’s native structure

TCA (acid)

Disrupts solvation layer of protein

Partially denatures protein exposing even more hydrophobic surface to solvent

Ionizing radiations

Stability of proteins with respect to denaturation is lowered upon treatment with ionizing radiations

Radiation break H-bonds



Factors affecting protein denaturation


Crosslinks tend to lower extent of denaturation or more difficult to unfold

Increased S-S linkages - increased stability, decreased denaturation


Disulphide bond (S-S)

Imidoester crosslinker dimethyl suberimidate

N-hydroxysuccinimide ester crosslinker BS3 and formaldehyde

Carbodiimide crosslinker - EDC

2 reasons :

When proteins are crosslinked it is more difficult for them to unfold

The more compact the molecule is and the greater the stability of protein

As energy is added to the system and secondary bonds are weakened, the presence of crosslinks will tend to maintain structure

This is especially true if the crosslinks are covalent as in the case of S-S bonds

While secondary forces may be weakened and some bonds can be broken, the crosslinkers

will tend to keep these groups in fairly close proximity

They also tend to prevent the exposure of large numbers of hydrophobic groups to the solvent

When conditions are returned to the native state, there is now a much greater chance for the

proper secondary interaction to occur and for the protein to assume the native configuration

If a protein can be caused to assume a completely random coil conformation, there will be a

large increase in entropy compared to the native structure

This entropy must be overcome if the protein is to refold into a native conformation

When crosslinking groups are present, a completely random coil conformation cannot be assumed

These groups introduce order into the structure and there is a considerable loss in the

amount of disorder that can be achieved in the most denatured state

Because of this, the entropy change between the native and denatured state is not nearly as great and there will be less of a driving force for denaturation If the crosslinking groups are broken before denaturation and thus allowed to randomly form after denaturation, no stability will be added to the protein by the pressure of these groups

Random coil disordered high entropy

Folded ordered low entropy

Protein Renaturation or Refolding

Denaturation may be reversible or irreversible

Reversible denaturation

The process of regaining normal protein properties by a denatured protein is called ‘renaturation’ or ‘refolding’

Renaturation occurs upon return of original conditions

Many cases have been observed in which an unfolded protein molecule spontaneously returns to its native biologically active form in test-tube

Denaturation is not irreversible (except when secondary and tertiary structures are completely lost)

Renaturation is possible in case of partial unfolding

Information of folding and refolding is in primary amino acid sequence

Some denatured proteins spontaneously recover their biological activity and thus their original native conformation, sometimes very rapidly

Proteins that spontaneously refold into their native conformation are stable than their denatured form under specific conditions of pH, ionic strength and temperature

Renaturation cannot evoke any biological activity that was not present in original protein

Proteins containing crosslinks refold spontaneously

If crosslinks are broken before denaturation, even then few proteins refold, e.g., RNase

Some proteins lacking S-S crosslinks can also refold into the ‘native’, active configuration spontaneously and quickly after denaturation

If the denatured protein is an enzyme, its catalytic activity returns on renaturation, without change in the specificity of the reaction catalyzed

Example, if trypsin is exposed to temperature of 80-90°C, it denatures and when the solution is cooled to 37°C the solubility and activity of this enzyme is regained

During renaturation, certain antibodies may cause re-rolling of protein bundles so that most of the original bonds are recovered

SRV 41

Irreversible denaturation

Proteins, when denatured, cannot be brought back to their original state; In that case, the

denaturation is ‘irreversible’

Unfolded form of some proteins may have less free energy than native form

In such cases, transition from native to random state may have a very high activation energy barrier thus locking the polypeptide chain into its native conformation

In this case, once the native form is unfolded, the polypeptide chain will not spontaneously refold into native conformation

Renaturation is not possible in case of complete unfolding or aggregation

It is assumed that the native conformation of a globular protein is more stable, i.e., has

less free energy, than the random coil form under biological conditions

But this assumption may not be true for all proteins Proteins that spontaneously refold (after denaturation) into their native form

May indeed be more stable (less energy) than their denatured forms under specific

conditions of pH, ionic strength and temperature

On the other hand, the unfolded proteins may have less free energy than the native form


this case, once

the native form is unfolded, the polypeptide

chain will not

spontaneously refold into the native conformation

At high temperature

Tertiary structure is lost


Protein refolds

Denaturation due to exposure to liquid- liquid interface and shear is essentially irreversible because of large energy barriers Even if phases are separated and protein is forced into aqueous phase, the protein will not regain its native original state Rather an association of hydrophobic groups will cause the protein to aggregate

Presence of S-S linkages help in

maintaining structure

S-S keeps secondary bond groups in close proximity

Prevents exposure of large number of hydrophobic groups to solvent

Upon return of conditions to native state there are greater chances of proper secondary interactions and protein assumes native state


High salt concentration

Decrease salt concentration or dilute


Recovery of native structure of protein

Water miscible organic solvent (less

polar than water)

Weakening of hydrophobic bonds

Change in protein structure

Protein refolds

Reversibility depends on

Nature of nonpolar solvent

Extent of unfolding

Temperature of system

Rate of solvent removal


Large amount of water miscible organic solvent EtOH or acetone

Protein largely unfolded with extensive exposure of hydrophobic groups

Protein when transferred to water at room temperature instantaneously

Protein aggregate and precipitate

Sudden exposure of hydrophobic groups to water → protein tries to remove hydrophobic groups from water → even before short range interactions could redirect folding, protein aggregation

would occur

Protein when transferred to water at room temperature slowly (exchange of solvent is slow)

There is better chance that hydrophobic groups would be able to

return to interior of molecule to prevent aggregation

If transferred to water and exchange of solvent is slow and at low temperature

Chances of regaining native structure is even better


To recover enzyme activity after enzyme purification step using organic solvent which is less polar and water miscible

Both solvent and protein solution should

be cold when they are mixed






performed at reduced temperatures