Common examplesWhen 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 in water. Fresh from the eggs, egg whites are transparent and liquid. Cooking the whites turns them opaque, forming an interconnected solid mass. The same transformation can be effected with a denaturing chemical. Pouring egg whites into a beaker of will also turn egg whites translucent and solid. The skin that forms on milk is another common example of denatured protein. The cold appetizer known as is prepared by chemically "cooking" raw fish and shellfish in an acidic citrus marinade, without heat.
Protein denaturationDenatured proteins can exhibit a wide range of characteristics, from loss of to .
Backgrounds or s are polymers of s. A protein is created by s that "read" RNA that is encoded by s in the gene and assemble the requisite amino acid combination from the instruction, in a process known as . The newly created protein strand then undergoes , in which additional s or s are added, for example , , or . Once this post-translational modification process has been completed, the protein begins to fold (sometimes spontaneously and sometimes with assistance), curling up on itself so that elements of the protein are buried deep inside the structure and 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. As a result, this process is heavily reliant on environmental state that the protein resides in. These environmental conditions include, and are not limited to, , , , and the solvents that happen to be involved. 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. When a protein is denatured, secondary and tertiary structures are altered but the s 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 , which are unfolded in their , but still functionally active and tend to fold upon binding to their biological target.
How denaturation occurs at levels of protein structure* 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: ** interactions between amino acid (such as s between groups) ** Non-covalent -dipole interactions between polar amino acid side-chains (and the surrounding ) ** between nonpolar amino acid side-chains. * In secondary structure denaturation, proteins lose all regular repeating patterns such as and , and adopt a configuration. * , such as the sequence of amino acids held together by covalent peptide bonds, is not disrupted by denaturation.
Loss of functionMost biological substrates lose their biological function when denatured. For example, s lose their , because the substrates can no longer bind to the , and because amino acid residues involved in stabilizing substrates' s 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 , , and .
Loss of activity due to heavy metals and metalloidsBy targeting proteins, heavy metals have been known to disrupt the function and activity carried out by proteins. It is important to note that heavy metals fall into categories consisting of transition metals as well as a select amount of metalloid. These metals, when interacting with native, folded proteins, tend to play a role in obstructing their biological activity. 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. Heavy metals also play a role in oxidizing amino acid side chains present in protein. Along with this, when interacting with metalloproteins, heavy metals can dislocate and replace key metal ions. As a result, heavy metals can interfere with folded proteins, which can strongly deter protein stability and activity.
Reversibility and irreversibilityIn many cases, denaturation is reversible (the proteins can regain their native state when the denaturing influence is removed). This process can be called renaturation. 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 that codes for the protein, the so-called " ". 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.
Protein denaturation due to pHDenaturation 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. Acid-induced unfolding often occurs between pH 2 and 5, base-induced unfolding usually requires pH 10 or higher.
Nucleic acid denaturations (including and ) are polymers synthesized by during either or . Following 5'-3' synthesis of the backbone, individual nitrogenous bases are capable of interacting with one another via ing, 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 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 "" 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 denaturationThe between in DNA can be broken in order to "open" the when biologically important mechanisms such as DNA replication, transcription, or protein binding are set to occur. 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. The first model that attempted to describe the 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 . As the temperature increases, the hydrogen bonds between the Watson and Crick base pairs are increasingly disturbed and "denatured loops" begin to form. However, the Poland-Scheraga Model is now considered elementary because it fails to account for the confounding implications of , chemical composition, and . Recent thermodynamic studies have inferred that the lifetime of a singular denaturation bubble ranges from 1 microsecond to 1 millisecond. This information is based on established timescales of DNA replication and transcription. Currently, biophysical and biochemical research studies are being performed to more fully elucidate the thermodynamic details of the denaturation bubble.
Denaturation due to chemical agentsWith (PCR) being among the most popular contexts in which DNA denaturation is desired, heating is the most frequent method of denaturation. Other than denaturation by heat, nucleic acids can undergo the denaturation process through various chemical agents such as , , , (DMSO), , and . These chemical denaturing agents lower the melting temperature (Tm) by competing for hydrogen bond donors and acceptors with pre-existing pairs. Some agents are even able to induce denaturation at room temperature. For example, agents (e.g. NaOH) have been shown to denature DNA by changing and removing hydrogen-bond contributing protons. These denaturants have been employed to make (DGGE), which promotes denaturation of nucleic acids in order to eliminate the influence of nucleic acid shape on their mobility.
Chemical denaturation as an alternativeThe (absorption and scattering of light) and hydrodynamic properties (, s, and s) of denatured nucleic acids are similar to those of heat-denatured nucleic acids. 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 , and chemical denaturation show that chemical denaturation can provide quicker denaturation compared to the other physical denaturation methods described. Particularly in cases where rapid renaturation is desired, chemical denaturation agents can provide an ideal alternative to heating. For example, DNA strands denatured with such as renature as soon as is added.
Denaturation due to airSmall, molecules such as and , which are the primary gases in , significantly impact the ability of surrounding molecules to participate in ing. 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. in DNA double helices are non-covalently bound by hydrogen bonding between Watson and Crick base pairs; nitrogen and oxygen therefore maintain the potential to weaken the integrity of DNA when exposed to air. As a result, DNA strands exposed to air require less force to separate and exemplify lower .
ApplicationsMany 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: * * * *
Acidsic protein denaturants include: * * 12% in water *
Baseswork similarly to acids in denaturation. They include: *
SolventsMost organic s are denaturing, including: *
Cross-linking reagentsing agents for proteins include: * *
Chaotropic agentss include: * 6 – 8 * 6 mol/l * 4.5 mol/l *
Disulfide bond reducersAgents that break s by reduction include: * * * (tris(2-carboxyethyl)phosphine)
Chemically reactive agentsAgents 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 * * Radiation * Temperature
Nucleic acid denaturants
Chemicalic nucleic acid denaturants include: * * HCl * Nitric Acid nucleic acid denaturants include: * NaOH Other nucleic acid denaturants include: * * * * * *
Physical* Thermal denaturation * Beads mill * Probe *
See also* * * * *