Denaturation (biochemistry)

In

Proper protein folding is key to whether a globular or membrane protein can do its job correctly; it must be folded into the native shape to function. However, hydrogen bonds and cofactor-protein binding, which play a crucial role in folding, are rather weak, and thus, easily affected by heat, acidity, varying salt concentrations, chelating agents, and other stressors which can denature the protein. This is one reason why cellular homeostasis is physiologically necessary in most life forms.

This concept is unrelated to denatured alcohol, which is alcohol that has been mixed with additives to make it unsuitable for human consumption.

Common examples

When food is cooked, some of its proteins become denatured. This is why boiled eggs become hard and cooked meat becomes firm.

A classic example of denaturing in proteins comes from egg whites, which are typically largely

Denatured proteins can exhibit a wide range of characteristics, from loss of solubility to protein aggregation.

Background

Protein folding consists of a balance between a substantial amount of weak intra-molecular interactions within a protein (Hydrophobic, electrostatic, and Van Der Waals Interactions) and protein-solvent interactions.^[7] As a result, this process is heavily reliant on environmental state that the protein resides in.^[7] These environmental conditions include, and are not limited to, temperature, salinity, pressure, and the solvents that happen to be involved.^[7] Consequently, any exposure to extreme stresses (e.g. heat or radiation, high inorganic salt concentrations, strong acids and bases) can disrupt a protein's interaction and inevitably lead to denaturation.^[8]

When a protein is denatured, secondary and tertiary structures are altered but the

• In quaternary structure denaturation, protein sub-units are dissociated and/or the spatial arrangement of protein subunits is disrupted.
• Tertiary structure denaturation involves the disruption of:
  • disulfide bridges between cysteine
    groups)
  • Non-covalent dipole-dipole interactions between polar amino acid side-chains (and the surrounding solvent)
  • Van der Waals (induced dipole) interactions between nonpolar amino acid side-chains.
• In secondary structure denaturation, proteins lose all regular repeating patterns such as alpha-helices and beta-pleated sheets, and adopt a random coil configuration.
• Primary structure, such as the sequence of amino acids held together by covalent peptide bonds, is not disrupted by denaturation.^[10]

Loss of function

Most biological substrates lose their biological function when denatured. For example, enzymes lose their activity, because the substrates can no longer bind to the active site,^[11] and because amino acid residues involved in stabilizing substrates' transition states are no longer positioned to be able to do so. The denaturing process and the associated loss of activity can be measured using techniques such as dual-polarization interferometry, CD, QCM-D and MP-SPR.

Loss of activity due to heavy metals and metalloids

By targeting proteins, heavy metals have been known to disrupt the function and activity carried out by proteins.^[12] It is important to note that heavy metals fall into categories consisting of transition metals as well as a select amount of metalloid.^[12] These metals, when interacting with native, folded proteins, tend to play a role in obstructing their biological activity.^[12] This interference can be carried out in a different number of ways. These heavy metals can form a complex with the functional side chain groups present in a protein or form bonds to free thiols.^[12] Heavy metals also play a role in oxidizing amino acid side chains present in protein.^[12] Along with this, when interacting with metalloproteins, heavy metals can dislocate and replace key metal ions.^[12] As a result, heavy metals can interfere with folded proteins, which can strongly deter protein stability and activity.

Reversibility and irreversibility

In many cases, denaturation is reversible (the proteins can regain their native state when the denaturing influence is removed). This process can be called renaturation.^[13] This understanding has led to the notion that all the information needed for proteins to assume their native state was encoded in the primary structure of the protein, and hence in the DNA that codes for the protein, the so-called "Anfinsen's thermodynamic hypothesis".^[14]

Denaturation can also be irreversible. This irreversibility is typically a kinetic, not thermodynamic irreversibility, as a folded protein generally has lower free energy than when it is unfolded. Through kinetic irreversibility, the fact that the protein is stuck in a local minimum can stop it from ever refolding after it has been irreversibly denatured.^[15]

Protein denaturation due to pH

Denaturation can also be caused by changes in the pH which can affect the chemistry of the amino acids and their residues. The ionizable groups in amino acids are able to become ionized when changes in pH occur. A pH change to more acidic or more basic conditions can induce unfolding.^[16] Acid-induced unfolding often occurs between pH 2 and 5, base-induced unfolding usually requires pH 10 or higher.^[16]

Nucleic acid denaturation

The

The first model that attempted to describe the thermodynamics of the denaturation bubble was introduced in 1966 and called the Poland-Scheraga Model. This model describes the denaturation of DNA strands as a function of temperature. As the temperature increases, the hydrogen bonds between the base pairs are increasingly disturbed and "denatured loops" begin to form.^[18] However, the Poland-Scheraga Model is now considered elementary because it fails to account for the confounding implications of DNA sequence, chemical composition, stiffness and torsion.^[19]

Recent thermodynamic studies have inferred that the lifetime of a singular denaturation bubble ranges from 1 microsecond to 1 millisecond.^[20] This information is based on established timescales of DNA replication and transcription.^[20] Currently,^[when?] biophysical and biochemical research studies are being performed to more fully elucidate the thermodynamic details of the denaturation bubble.^[20]

Denaturation due to chemical agents

With

The optical activity (absorption and scattering of light) and hydrodynamic properties (translational diffusion, sedimentation coefficients, and rotational correlation times) of formamide denatured nucleic acids are similar to those of heat-denatured nucleic acids.^[22][24][25] Therefore, depending on the desired effect, chemically denaturing DNA can provide a gentler procedure for denaturing nucleic acids than denaturation induced by heat. Studies comparing different denaturation methods such as heating, beads mill of different bead sizes, probe sonication, and chemical denaturation show that chemical denaturation can provide quicker denaturation compared to the other physical denaturation methods described.^[21] Particularly in cases where rapid renaturation is desired, chemical denaturation agents can provide an ideal alternative to heating. For example, DNA strands denatured with alkaline agents such as NaOH renature as soon as phosphate buffer is added.^[21]

Denaturation due to air

Small, electronegative molecules such as nitrogen and oxygen, which are the primary gases in air, significantly impact the ability of surrounding molecules to participate in hydrogen bonding.^[26] These molecules compete with surrounding hydrogen bond acceptors for hydrogen bond donors, therefore acting as "hydrogen bond breakers" and weakening interactions between surrounding molecules in the environment.^[26] Antiparellel strands in DNA double helices are non-covalently bound by hydrogen bonding between base pairs;^[27] nitrogen and oxygen therefore maintain the potential to weaken the integrity of DNA when exposed to air.^[28] As a result, DNA strands exposed to air require less force to separate and exemplify lower melting temperatures.^[28]

Applications

Many laboratory techniques rely on the ability of nucleic acid strands to separate. By understanding the properties of nucleic acid denaturation, the following methods were created:

• PCR
• Southern blot
• Northern blot
• DNA sequencing

Denaturants

Protein denaturants

Acids

Acidic protein denaturants include:

• Acetic acid^[29]
• Trichloroacetic acid 12% in water
• Sulfosalicylic acid

Bases

Bases work similarly to acids in denaturation. They include:

• Sodium bicarbonate

Solvents

Most organic solvents are denaturing, including:^{[citation needed]}

• Ethanol

Cross-linking reagents

Cross-linking agents for proteins include:^{[citation needed]}

• Formaldehyde
• Glutaraldehyde

Chaotropic agents

Chaotropic agents include:^{[citation needed]}

• mol/L
• Guanidinium chloride 6 mol/L
• Lithium perchlorate 4.5 mol/L
• Sodium dodecyl sulfate

Disulfide bond reducers

Agents that break

• 2-Mercaptoethanol
• Dithiothreitol
• TCEP (tris(2-carboxyethyl)phosphine)

Chemically reactive agents

Agents such as hydrogen peroxide, elemental chlorine, hypochlorous acid (chlorine water), bromine, bromine water, iodine, nitric and oxidising acids, and ozone react with sensitive moieties such as sulfide/thiol, activated aromatic rings (phenylalanine) in effect damage the protein and render it useless.

Other

• Mechanical agitation
• Picric acid
• Radiation
• Temperature^[30]

Nucleic acid denaturants

Chemical

Acidic nucleic acid denaturants include:

• Acetic acid
• HCl
• Nitric acid

Basic nucleic acid denaturants include:

• NaOH

Other nucleic acid denaturants include:

• DMSO
• Formamide
• Guanidine
• Sodium salicylate
• Propylene glycol
• Urea

Physical

• Thermal denaturation
• Beads mill
• Probe sonication
• Radiation

See also

• Denatured alcohol
• Equilibrium unfolding
• Fixation (histology)
• Protein folding
• Random coil

References