Denaturation (biochemistry)

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The effects of temperature on enzyme activity. Top: increasing temperature increases the rate of reaction (Q10 coefficient). Middle: the fraction of folded and functional enzyme decreases above its denaturation temperature. Bottom: consequently, an enzyme's optimal rate of reaction is at an intermediate temperature. Q10 graphs.svg
The effects of temperature on enzyme activity.
Top: increasing temperature increases the rate of reaction (Q10 coefficient).
Middle: the fraction of folded and functional enzyme decreases above its denaturation temperature.
Bottom: consequently, an enzyme's optimal rate of reaction is at an intermediate temperature.
IUPAC definition for denaturation (of a macromolecule) IUPAC definition for denaturation (of a macromolecule).png
IUPAC definition for denaturation (of a macromolecule)

In biochemistry, denaturation is a process in which proteins or nucleic acids lose the quaternary structure, tertiary structure, and secondary structure which is present in their native state, by application of some external stress or compound, such as a strong acid or base, a concentrated inorganic salt, an organic solvent (e.g., alcohol or chloroform), agitation and radiation, or heat. [1] If proteins in a living cell are denatured, this results in disruption of cell activity and possibly cell death. Protein denaturation is also a consequence of cell death. [2] [3] Denatured proteins can exhibit a wide range of characteristics, from conformational change and loss of solubility to aggregation due to the exposure of hydrophobic groups. The loss of solubility as a result of denaturation is called coagulation. [4] Denatured proteins lose their 3D structure, and therefore, cannot function.

Contents

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, which play a big part in folding, are rather weak, and thus, easily affected by heat, acidity, varying salt concentrations, and other stressors which can denature the protein. This is one reason why cellular homeostasis is physiologically necessary in many 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

(Top) The protein albumin in the egg white undergoes denaturation and loss of solubility when the egg is cooked. (Bottom) Paperclips provide a visual analogy to help with the conceptualization of the denaturation process. Protein Denaturation.png
(Top) The protein albumin in the egg white undergoes denaturation and loss of solubility when the egg is cooked. (Bottom) Paperclips provide a visual analogy to help with the conceptualization of the denaturation process.

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 egg albumins in water. Fresh from the eggs, egg whites are transparent and liquid. Cooking the thermally unstable whites turns them opaque, forming an interconnected solid mass. [5] The same transformation can be effected with a denaturing chemical. Pouring egg whites into a beaker of acetone will also turn egg whites translucent and solid. The skin that forms on curdled milk is another common example of denatured protein. The cold appetizer known as ceviche is prepared by chemically "cooking" raw fish and shellfish in an acidic citrus marinade, without heat. [6]

Protein denaturation

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

Functional proteins have four levels of structural organization: Primary structure: the linear structure of amino acids in the polypeptide chain Secondary structure: hydrogen bonds between peptide group chains in an alpha helix or beta sheet Tertiary structure: three-dimensional structure of alpha helixes and beta helixes folded Quaternary structure: three-dimensional structure of multiple polypeptides and how they fit together Levels of structural organization of a protein.svg
Functional proteins have four levels of structural organization:
  1. Primary structure: the linear structure of amino acids in the polypeptide chain
  2. Secondary structure: hydrogen bonds between peptide group chains in an alpha helix or beta sheet
  3. Tertiary structure: three-dimensional structure of alpha helixes and beta helixes folded
  4. Quaternary structure: three-dimensional structure of multiple polypeptides and how they fit together
Process of denaturation: Functional protein showing a quaternary structure When heat is applied it alters the intramolecular bonds of the protein Unfolding of the polypeptides (amino acids) Process of Denaturation.svg
Process of denaturation:
  1. Functional protein showing a quaternary structure
  2. When heat is applied it alters the intramolecular bonds of the protein
  3. Unfolding of the polypeptides (amino acids)

Background

Proteins or polypeptides are polymers of amino acids. A protein is created by ribosomes that "read" RNA that is encoded by codons in the gene and assemble the requisite amino acid combination from the genetic instruction, in a process known as translation. The newly created protein strand then undergoes posttranslational modification, in which additional atoms or molecules are added, for example copper, zinc, or iron. Once this post-translational modification process has been completed, the protein begins to fold (sometimes spontaneously and sometimes with enzymatic assistance), curling up on itself so that hydrophobic elements of the protein are buried deep inside the structure and hydrophilic elements end up on the outside. The final shape of a protein determines how it interacts with its environment.

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 peptide bonds of the primary structure between the amino acids are left intact. Since all structural levels of the protein determine its function, the protein can no longer perform its function once it has been denatured. This is in contrast to intrinsically unstructured proteins, which are unfolded in their native state, but still functionally active and tend to fold upon binding to their biological target. [9]

How denaturation occurs at levels of protein structure

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

Nucleic acids (including RNA and DNA) are nucleotide polymers synthesized by polymerase enzymes during either transcription or DNA replication. Following 5'-3' synthesis of the backbone, individual nitrogenous bases are capable of interacting with one another via hydrogen bonding, thus allowing for the formation of higher-order structures. Nucleic acid denaturation occurs when hydrogen bonding between nucleotides is disrupted, and results in the separation of previously annealed strands. For example, denaturation of DNA due to high temperatures results in the disruption of base pairs and the separation of the double stranded helix into two single strands. Nucleic acid strands are capable of re-annealling when "normal" conditions are restored, but if restoration occurs too quickly, the nucleic acid strands may re-anneal imperfectly resulting in the improper pairing of bases.

Biologically-induced denaturation

DNA denaturation occurs when hydrogen bonds between base pairs are disturbed. DNA Denaturation.png
DNA denaturation occurs when hydrogen bonds between base pairs are disturbed.

The non-covalent interactions between antiparallel strands in DNA can be broken in order to "open" the double helix when biologically important mechanisms such as DNA replication, transcription, DNA repair or protein binding are set to occur. [17] The area of partially separated DNA is known as the denaturation bubble, which can be more specifically defined as the opening of a DNA double helix through the coordinated separation of base pairs. [17]

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

Formamide denatures DNA by disrupting the hydrogen bonds between base pairs. Orange, blue, green, and purple lines represent adenine, thymine, guanine, and cytosine respectively. The three short black lines between the bases and the formamide molecules represent newly formed hydrogen bonds. DNA Denaturation by Formamide.png
Formamide denatures DNA by disrupting the hydrogen bonds between base pairs. Orange, blue, green, and purple lines represent adenine, thymine, guanine, and cytosine respectively. The three short black lines between the bases and the formamide molecules represent newly formed hydrogen bonds.

With polymerase chain reaction (PCR) being among the most popular contexts in which DNA denaturation is desired, heating is the most frequent method of denaturation. [21] Other than denaturation by heat, nucleic acids can undergo the denaturation process through various chemical agents such as formamide, guanidine, sodium salicylate, dimethyl sulfoxide (DMSO), propylene glycol, and urea. [22] These chemical denaturing agents lower the melting temperature (Tm) by competing for hydrogen bond donors and acceptors with pre-existing nitrogenous base pairs. Some agents are even able to induce denaturation at room temperature. For example, alkaline agents (e.g. NaOH) have been shown to denature DNA by changing pH and removing hydrogen-bond contributing protons. [21] These denaturants have been employed to make Denaturing Gradient Gel Electrophoresis gel (DGGE), which promotes denaturation of nucleic acids in order to eliminate the influence of nucleic acid shape on their electrophoretic mobility. [23]

Chemical denaturation as an alternative

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:

Denaturants

Protein denaturants

Acids

Acidic protein denaturants include:

Bases

Bases work similarly to acids in denaturation. They include:

Solvents

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

Cross-linking reagents

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

Chaotropic agents

Chaotropic agents include:[ citation needed ]

Disulfide bond reducers

Agents that break disulfide bonds by reduction include:[ citation needed ]

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

Nucleic acid denaturants

Chemical

Acidic nucleic acid denaturants include:

Basic nucleic acid denaturants include:

  • NaOH

Other nucleic acid denaturants include:

Physical

See also

Related Research Articles

<span class="mw-page-title-main">Alpha helix</span> Type of secondary structure of proteins

An alpha helix is a sequence of amino acids in a protein that are twisted into a coil.

<span class="mw-page-title-main">Base pair</span> Unit consisting of two nucleobases bound to each other by hydrogen bonds

A base pair (bp) is a fundamental unit of double-stranded nucleic acids consisting of two nucleobases bound to each other by hydrogen bonds. They form the building blocks of the DNA double helix and contribute to the folded structure of both DNA and RNA. Dictated by specific hydrogen bonding patterns, "Watson–Crick" base pairs allow the DNA helix to maintain a regular helical structure that is subtly dependent on its nucleotide sequence. The complementary nature of this based-paired structure provides a redundant copy of the genetic information encoded within each strand of DNA. The regular structure and data redundancy provided by the DNA double helix make DNA well suited to the storage of genetic information, while base-pairing between DNA and incoming nucleotides provides the mechanism through which DNA polymerase replicates DNA and RNA polymerase transcribes DNA into RNA. Many DNA-binding proteins can recognize specific base-pairing patterns that identify particular regulatory regions of genes.

<span class="mw-page-title-main">Gel electrophoresis</span> Method for separation and analysis of biomolecules

Gel electrophoresis is a method for separation and analysis of biomacromolecules and their fragments, based on their size and charge. It is used in clinical chemistry to separate proteins by charge or size and in biochemistry and molecular biology to separate a mixed population of DNA and RNA fragments by length, to estimate the size of DNA and RNA fragments or to separate proteins by charge.

<span class="mw-page-title-main">Protein biosynthesis</span> Assembly of proteins inside biological cells

Protein biosynthesis is a core biological process, occurring inside cells, balancing the loss of cellular proteins through the production of new proteins. Proteins perform a number of critical functions as enzymes, structural proteins or hormones. Protein synthesis is a very similar process for both prokaryotes and eukaryotes but there are some distinct differences.

<span class="mw-page-title-main">Protein folding</span> Change of a linear protein chain to a 3D structure

Protein folding is the physical process by which a protein, after synthesis by a ribosome as a linear chain of amino acids, changes from an unstable random coil into a more ordered three-dimensional structure. This structure permits the protein to become biologically functional.

<span class="mw-page-title-main">Active site</span> Active region of an enzyme

In biology and biochemistry, the active site is the region of an enzyme where substrate molecules bind and undergo a chemical reaction. The active site consists of amino acid residues that form temporary bonds with the substrate, the binding site, and residues that catalyse a reaction of that substrate, the catalytic site. Although the active site occupies only ~10–20% of the volume of an enzyme, it is the most important part as it directly catalyzes the chemical reaction. It usually consists of three to four amino acids, while other amino acids within the protein are required to maintain the tertiary structure of the enzymes.

In biochemistry, the native state of a protein or nucleic acid is its properly folded and/or assembled form, which is operative and functional. The native state of a biomolecule may possess all four levels of biomolecular structure, with the secondary through quaternary structure being formed from weak interactions along the covalently-bonded backbone. This is in contrast to the denatured state, in which these weak interactions are disrupted, leading to the loss of these forms of structure and retaining only the biomolecule's primary structure.

<span class="mw-page-title-main">Protein structure</span> Three-dimensional arrangement of atoms in an amino acid-chain molecule

Protein structure is the three-dimensional arrangement of atoms in an amino acid-chain molecule. Proteins are polymers – specifically polypeptides – formed from sequences of amino acids, which are the monomers of the polymer. A single amino acid monomer may also be called a residue, which indicates a repeating unit of a polymer. Proteins form by amino acids undergoing condensation reactions, in which the amino acids lose one water molecule per reaction in order to attach to one another with a peptide bond. By convention, a chain under 30 amino acids is often identified as a peptide, rather than a protein. To be able to perform their biological function, proteins fold into one or more specific spatial conformations driven by a number of non-covalent interactions, such as hydrogen bonding, ionic interactions, Van der Waals forces, and hydrophobic packing. To understand the functions of proteins at a molecular level, it is often necessary to determine their three-dimensional structure. This is the topic of the scientific field of structural biology, which employs techniques such as X-ray crystallography, NMR spectroscopy, cryo-electron microscopy (cryo-EM) and dual polarisation interferometry, to determine the structure of proteins.

A chaotropic agent is a molecule in water solution that can disrupt the hydrogen bonding network between water molecules. This has an effect on the stability of the native state of other molecules in the solution, mainly macromolecules by weakening the hydrophobic effect. For example, a chaotropic agent reduces the amount of order in the structure of a protein formed by water molecules, both in the bulk and the hydration shells around hydrophobic amino acids, and may cause its denaturation.

<span class="mw-page-title-main">Hydrophobic effect</span> Aggregation of non-polar molecules in aqueous solutions

The hydrophobic effect is the observed tendency of nonpolar substances to aggregate in an aqueous solution and to be excluded by water. The word hydrophobic literally means "water-fearing", and it describes the segregation of water and nonpolar substances, which maximizes the entropy of water and minimizes the area of contact between water and nonpolar molecules. In terms of thermodynamics, the hydrophobic effect is the free energy change of water surrounding a solute. A positive free energy change of the surrounding solvent indicates hydrophobicity, whereas a negative free energy change implies hydrophilicity.

In chemistry, a non-covalent interaction differs from a covalent bond in that it does not involve the sharing of electrons, but rather involves more dispersed variations of electromagnetic interactions between molecules or within a molecule. The chemical energy released in the formation of non-covalent interactions is typically on the order of 1–5 kcal/mol. Non-covalent interactions can be classified into different categories, such as electrostatic, π-effects, van der Waals forces, and hydrophobic effects.

<span class="mw-page-title-main">Thermostability</span> Ability of a substance to resist changes in structure under high temperatures

In materials science and molecular biology, thermostability is the ability of a substance to resist irreversible change in its chemical or physical structure, often by resisting decomposition or polymerization, at a high relative temperature.

<span class="mw-page-title-main">Beta hairpin</span>

The beta hairpin is a simple protein structural motif involving two beta strands that look like a hairpin. The motif consists of two strands that are adjacent in primary structure, oriented in an antiparallel direction, and linked by a short loop of two to five amino acids. Beta hairpins can occur in isolation or as part of a series of hydrogen bonded strands that collectively comprise a beta sheet.

<span class="mw-page-title-main">Hyperchromicity</span>

Hyperchromicity is the increase of absorbance of a material. The most famous example is the hyperchromicity of DNA that occurs when the DNA duplex is denatured. The UV absorption is increased when the two single DNA strands are being separated, either by heat or by addition of denaturant or by increasing the pH level. The opposite, a decrease of absorbance is called hypochromicity.

<span class="mw-page-title-main">Cyclol</span> Structural model of a folded, globular protein

The cyclol hypothesis is the now discredited first structural model of a folded, globular protein, formulated in the 1930s. It was based on the cyclol reaction of peptide bonds proposed by physicist Frederick Frank in 1936, in which two peptide groups are chemically crosslinked. These crosslinks are covalent analogs of the non-covalent hydrogen bonds between peptide groups and have been observed in rare cases, such as the ergopeptides.

Protein metabolism denotes the various biochemical processes responsible for the synthesis of proteins and amino acids (anabolism), and the breakdown of proteins by catabolism.

Nucleic acid thermodynamics is the study of how temperature affects the nucleic acid structure of double-stranded DNA (dsDNA). The melting temperature (Tm) is defined as the temperature at which half of the DNA strands are in the random coil or single-stranded (ssDNA) state. Tm depends on the length of the DNA molecule and its specific nucleotide sequence. DNA, when in a state where its two strands are dissociated, is referred to as having been denatured by the high temperature.

<span class="mw-page-title-main">Proteinase K</span> Broad-spectrum serine protease

In molecular biology, Proteinase K is a broad-spectrum serine protease. The enzyme was discovered in 1974 in extracts of the fungus Parengyodontium album. Proteinase K is able to digest hair (keratin), hence, the name "Proteinase K". The predominant site of cleavage is the peptide bond adjacent to the carboxyl group of aliphatic and aromatic amino acids with blocked alpha amino groups. It is commonly used for its broad specificity. This enzyme belongs to Peptidase family S8 (subtilisin). The molecular weight of Proteinase K is 28,900 daltons.

<span class="mw-page-title-main">Nucleic acid structure</span> Biomolecular structure of nucleic acids such as DNA and RNA

Nucleic acid structure refers to the structure of nucleic acids such as DNA and RNA. Chemically speaking, DNA and RNA are very similar. Nucleic acid structure is often divided into four different levels: primary, secondary, tertiary, and quaternary.

<span class="mw-page-title-main">Nucleic acid secondary structure</span>

Nucleic acid secondary structure is the basepairing interactions within a single nucleic acid polymer or between two polymers. It can be represented as a list of bases which are paired in a nucleic acid molecule. The secondary structures of biological DNAs and RNAs tend to be different: biological DNA mostly exists as fully base paired double helices, while biological RNA is single stranded and often forms complex and intricate base-pairing interactions due to its increased ability to form hydrogen bonds stemming from the extra hydroxyl group in the ribose sugar.

References

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