Protein Denaturation and Function

Protein denaturation is the loss of the native three-dimensional structure of a protein, caused by heat, extreme pH, organic solvents, detergents or heavy metals, and it destroys the function of the protein while leaving the peptide bonds and the amino acid sequence intact. In brief: cooking an egg denatures the proteins of the egg white at about 62–85 °C, and the process is irreversible because the unfolded proteins aggregate; most human enzymes work best near 37 °C and lose activity quickly above about 45 °C.

The function of a protein depends on a precise shape that is held by weak forces, which are easy to disrupt. Denaturation explains why food is cooked, why milk curdles, why fever above 41 °C is dangerous and why sterilization with heat or alcohol works. This article describes the causes of denaturation with a table, shows how the structure relates to the function using sickle-cell hemoglobin and enzymes, gives calculations of the denaturation rate and the melting temperature, and discusses refolding, chaperones and misfolding diseases.

What is denaturation?

A native protein is folded into a specific shape that is held together mainly by non-covalent interactions: hydrogen bonds, ionic bonds, hydrophobic interactions and van der Waals forces, and sometimes disulfide bonds. The stability of the folded state is small: the difference in free energy between the folded and unfolded form is only about 20–60 kJ/mol, which is equivalent to a few hydrogen bonds. When these interactions are disrupted, the protein unfolds. The secondary, tertiary and quaternary structures are lost, but the primary structure is not changed, since the covalent peptide bonds are not broken by the mild conditions that cause denaturation. The unfolded protein usually loses its biological activity, and its solubility drops, so it often forms a precipitate or coagulate.

What causes denaturation?

• Heat: increases the motion of atoms and breaks hydrogen bonds and hydrophobic contacts.
• Extreme pH: acids or bases change the charge of the side chains, break ionic bonds and cause repulsion.
• Organic solvents (ethanol, acetone): disrupt hydrophobic interactions and compete for hydrogen bonds.
• Chaotropic agents (urea at 6–8 M, guanidinium chloride): weaken the hydrophobic effect and hydrogen bonds.
• Detergents (sodium dodecyl sulfate, SDS): bind to the protein and unfold it, and coat it with negative charges.
• Heavy metal ions (Pb²⁺, Hg²⁺, Ag⁺): bind to –SH groups and carboxylates, and break salt bridges and disulfide bonds.
• Reducing agents (mercaptoethanol): break disulfide bonds.
• Mechanical agitation: whipping egg white unfolds proteins at the air–water interface.

Denaturing agents and their effects

Is denaturation reversible?

Sometimes. Anfinsen showed that ribonuclease A, unfolded with urea and mercaptoethanol, refolds into the active enzyme when the agents are removed slowly, which proved that the amino acid sequence determines the structure. Such renaturation is possible for small proteins in dilute solutions. For most proteins, especially after heating, denaturation is irreversible: the unfolded chains expose hydrophobic regions, which stick to those of other molecules and form aggregates, as in a cooked egg. In living cells, chaperones (such as the heat-shock proteins Hsp70 and the chaperonin GroEL) bind unfolded proteins, prevent aggregation and help them to fold. The cell produces more heat-shock proteins after a rise in temperature.

How do structure and function relate?

The shape of a protein creates the binding sites and the chemical environments that give it function. If the shape is lost, so is the function:

• Enzymes: the active site is formed by a few amino acids brought together by the fold. A denatured enzyme is inactive. A change of the shape slightly can also reduce activity.
• Hemoglobin: the replacement of a single glutamic acid by valine in the β chain gives hemoglobin S, which in the deoxygenated state polymerizes into fibers and deforms the red blood cell into a sickle shape that blocks capillaries.
• Antibodies: the shape of the binding site decides which antigen is recognized.
• Collagen: the triple helix gives strength to skin and tendons; heating in water converts it to gelatin.
• Prions: a misfolded form of a normal brain protein can induce other molecules to take the same shape, which causes diseases such as Creutzfeldt–Jakob disease. Prions are very resistant to the usual sterilization.

How do temperature and pH affect enzymes?

Enzyme activity rises with temperature, since the rate constant increases (by a factor of about 2 for every 10 °C), up to an optimum, and then falls sharply as denaturation takes over. Most human enzymes have an optimum near 37 °C. Enzymes of thermophilic bacteria, such as Taq polymerase from Thermus aquaticus, remain active at 72 °C and even survive 95 °C, which is the reason why they are used in the polymerase chain reaction (PCR). Each enzyme also has an optimum pH, which reflects its environment: pepsin in the stomach (pH 1.5–2), salivary amylase near 6.8 and trypsin in the small intestine at about 8.

Worked examples

Example 1: first-order loss of activity

An enzyme is held at 60 °C and loses its activity as a first-order process with k = 0.0231 min⁻¹. The half-life is t_½ = 0.693 / 0.0231 = 30.0 min. After 90 min, the remaining activity is (½)³ = 12.5 %.

Example 2: the melting temperature

The midpoint of the unfolding transition is called the melting temperature T_m. For a protein with T_m = 65 °C, half the molecules are unfolded at 65 °C. A mutation that raises T_m to 72 °C makes the protein more stable. Two-state unfolding can be described by K = [U]/[N] and ΔG = −RT ln K: at T_m, K = 1 and ΔG = 0. At 25 °C, a protein with ΔG_unfolding = +40 kJ/mol has K = e^−40/2.479 = 1 × 10⁻⁷, meaning that only 1 molecule in 10 million is unfolded at a given time.

Example 3: pasteurization and sterilization

Milk is pasteurized at about 72 °C for 15 s. This is enough to denature the proteins of most pathogens and spoilage organisms, but it denatures only a small fraction of milk proteins, and so milk keeps its taste. An autoclave sterilizes with steam at 121 °C for 15–20 min, which denatures even the very heat-resistant proteins of bacterial spores.

Example 4: the temperature of cooking eggs

The proteins of egg white denature at different temperatures: ovotransferrin at about 62 °C, ovalbumin (the most abundant, 54 %) at about 80–84 °C, and the egg yolk proteins set at about 65–70 °C. This is why a “soft-boiled” egg at 65 °C has a set white and a runny yolk, and a hard-boiled egg at 100 °C is fully set.

Example 5: protein precipitation by acid

Casein, the main protein in milk, has an isoelectric point of 4.6. When bacteria produce lactic acid in the making of yogurt and the pH falls to about 4.6, the net charge on casein goes to zero, the repulsion between the molecules disappears and they aggregate, and so the milk thickens or curdles.

How was denaturation studied?

The coagulation of egg white by heat was known from ancient times. Hsien Wu proposed in 1931 that denaturation is the unfolding of a protein molecule, and Alfred Mirsky and Linus Pauling developed this idea in 1936 and described the native structure as held together by hydrogen bonds. Christian Anfinsen’s work on ribonuclease (1961) showed that the native structure is determined by the sequence, and that denaturation can be reversible. The theory that proteins are only marginally stable was established by Charles Tanford and others, who studied denaturation with urea and guanidinium chloride in the 1960s.

Which mistakes are common?

• Saying that denaturation breaks peptide bonds: it does not; the sequence stays the same.
• Thinking that denatured proteins are always dead or useless as food: cooking makes many proteins easier to digest.
• Assuming that all denaturation is irreversible: some small proteins refold.
• Forgetting the role of pH: acids and bases denature proteins without heat.
• Mixing up the activity loss by denaturation and the reversible slowing at low temperature: a cold enzyme is slow but intact.