Process of partial or total alteration of the native secondary, and/or tertiary, and/or quaternary structures of proteins or nucleic acids resulting in a loss ofbioactivity.
Note 1: Modified from the definition given in ref.[1]
Note 2: Denaturation can occur when proteins and nucleic acids are subjected to elevated temperature or to extremes of pH, or to nonphysiological concentrations of salt, organic solvents, urea, or other chemical agents.
Note 3: Anenzyme loses its ability to alter or speed up a chemical reaction when it is denaturized.[2]
Inbiochemistry,denaturation is a process in whichproteins ornucleic acids losefolded structure present in theirnative state due to various factors, including application of some external stress or compound, such as a strongacid orbase, a concentratedinorganic salt, anorganic solvent (e.g.,alcohol orchloroform), agitation, radiation, orheat.[3] If proteins in a livingcell are denatured, this results in disruption of cell activity and possiblycell death. Protein denaturation is also a consequence of cell death.[4][5] Denatured proteins can exhibit a wide range of characteristics, fromconformational change and loss ofsolubility or dissociation ofcofactors toaggregation due to the exposure ofhydrophobic groups. The loss of solubility as a result of denaturation is calledcoagulation.[6] When denatured, proteins, e.g.,metalloenzymes, lose their3D structure or metal cofactor and, therefore, cannot function.
Properprotein folding is key to whether aglobular ormembrane 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 cellularhomeostasis isphysiologically necessary in mostlife forms.
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 largelyegg albumins in water. Fresh from the eggs, egg whites are transparent and liquid. Cooking thethermally unstable whites turns them opaque, forming an interconnected solid mass.[7] The same transformation can be effected with a denaturing chemical. Pouring egg whites into a beaker ofacetone will also turn egg whitestranslucent and solid. The skin that forms oncurdled milk is another common example of denatured protein. The cold appetizer known asceviche is prepared by chemically "cooking" raw fish and shellfish in an acidic citrus marinade, without heat.[8]
Denatured proteins can exhibit a wide range of characteristics, from loss ofsolubility toprotein aggregation.
Proteins orpolypeptides are polymers ofamino acids. A protein is created byribosomes that "read" RNA that is encoded bycodons in the gene and assemble the requisite amino acid combination from thegenetic instruction, in a process known astranslation. The newly created protein strand then undergoesposttranslational modification, in which additionalatoms ormolecules are added, for examplecopper,zinc, oriron. Once this post-translational modification process has been completed, the protein begins to fold (sometimes spontaneously and sometimes withenzymatic assistance), curling up on itself so thathydrophobic elements of the protein are buried deep inside the structure andhydrophilic 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.[9] As a result, this process is heavily reliant on environmental state that the protein resides in.[9] These environmental conditions include, and are not limited to,temperature,salinity,pressure, and the solvents that happen to be involved.[9] 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.[10]
When a protein is denatured, secondary and tertiary structures are altered but thepeptide 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 tointrinsically unstructured proteins, which are unfolded in theirnative state, but still functionally active and tend to fold upon binding to their biological target.[11]
Most biological substrates lose their biological function when denatured. For example,enzymes lose theiractivity, because the substrates can no longer bind to theactive site,[13] 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 asdual-polarization interferometry,CD,QCM-D andMP-SPR.
By targeting proteins, heavy metals have been known to disrupt the function and activity carried out by proteins.[14] Heavy metals fall into categories consisting of transition metals as well as a select amount ofmetalloid.[14] These metals, when interacting with native, folded proteins, tend to play a role in obstructing their biological activity.[14] 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.[14] Heavy metals also play a role in oxidizing amino acid side chains present in protein.[14] Along with this, when interacting with metalloproteins, heavy metals can dislocate and replace key metal ions.[14] As a result, heavy metals can interfere with folded proteins, which can strongly deter protein stability and activity.
In many cases, denaturation is reversible (the proteins can regain their native state when the denaturing influence is removed). This process can be calledrenaturation.[15] 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 theDNA that codes for the protein, the so-called "Anfinsen'sthermodynamic hypothesis".[16]
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.[17]
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.[18] Acid-induced unfolding often occurs between pH 2 and 5, base-induced unfolding usually requires pH 10 or higher.[18]
When proteins are folded, they fold so as to keep theirhydrophobic parts on the inside (away from water) and theirhydrophilic parts on the outside (contacting the water). This makes them soluble enough not to precipitate. However, when denatured the surface of the protein is partly hydrophobic and partly hydrophilic (as it no longer has an "inside" in which to hide the hydrophobic parts), causing it to become insoluble in water. The hydrophobic parts of the denatured proteins stick together, forming a network (gel): this is calledcoagulation.[19]
The coagulation of denatured proteins is the reasoneggs solidify when cooked.[19] When acid is added to milk, the proteincasein denatures and coagulates (with fat and water inclusions from the milk) intocurds, the first step in makingcheese;[19] although milk can also be made to curdle (i.e. casein to coagulate) by other methods, for example the addition of enzymes likechymosin.[20]
Nucleic acids (includingRNA andDNA) arenucleotide polymers synthesized bypolymerase enzymes during eithertranscription orDNA replication. Following 5'-3' synthesis of the backbone, individualnitrogenous bases are capable of interacting with one another viahydrogen 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 previouslyannealed strands. For example, denaturation of DNA due to high temperatures results in the disruption ofbase 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.
Thenon-covalent interactions betweenantiparallel strands in DNA can be broken in order to "open" thedouble helix when biologically important mechanisms such as DNA replication, transcription,DNA repair or protein binding are set to occur.[21] 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.[21]
The first model that attempted to describe thethermodynamics 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 oftemperature. As the temperature increases, the hydrogen bonds between the base pairs are increasingly disturbed and "denatured loops" begin to form.[22] However, the Poland-Scheraga Model is now considered elementary because it fails to account for the confounding implications ofDNA sequence, chemical composition,stiffness andtorsion.[23]
Recent thermodynamic studies have inferred that the lifetime of a singular denaturation bubble ranges from 1 microsecond to 1 millisecond.[24] This information is based on established timescales of DNA replication and transcription.[24] Currently,[when?] biophysical and biochemical research studies are being performed to more fully elucidate the thermodynamic details of the denaturation bubble.[24]
Withpolymerase chain reaction (PCR) being among the most popular contexts in which DNA denaturation is desired, heating is the most frequent method of denaturation.[25] Other than denaturation by heat, nucleic acids can undergo the denaturation process through various chemical agents such asformamide,guanidine,sodium salicylate,dimethyl sulfoxide (DMSO),propylene glycol, andurea.[26] These chemical denaturing agents lower the melting temperature (Tm) by competing for hydrogen bond donors and acceptors with pre-existingnitrogenous 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 changingpH and removing hydrogen-bond contributing protons.[25] These denaturants have been employed to makeDenaturing Gradient Gel Electrophoresis gel (DGGE), which promotes denaturation of nucleic acids in order to eliminate the influence of nucleic acid shape on theirelectrophoretic mobility.[27]
Theoptical activity (absorption and scattering of light) and hydrodynamic properties (translational diffusion,sedimentation coefficients, androtational correlation times) offormamide denatured nucleic acids are similar to those of heat-denatured nucleic acids.[26][28][29] 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, probesonication, and chemical denaturation show that chemical denaturation can provide quicker denaturation compared to the other physical denaturation methods described.[25] Particularly in cases where rapid renaturation is desired, chemical denaturation agents can provide an ideal alternative to heating. For example, DNA strands denatured withalkaline agents such asNaOH renature as soon asphosphate buffer is added.[25]
Small,electronegative molecules such asnitrogen andoxygen, which are the primary gases inair, significantly impact the ability of surrounding molecules to participate inhydrogen bonding.[30] 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.[30]Antiparellel strands in DNA double helices are non-covalently bound by hydrogen bonding between base pairs;[31] nitrogen and oxygen therefore maintain the potential to weaken the integrity of DNA when exposed to air.[32] As a result, DNA strands exposed to air require less force to separate and exemplify lowermelting temperatures.[32]
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:
Acidic protein denaturants include:
Bases work similarly to acids in denaturation. They include:
Most organicsolvents are denaturing, including:[citation needed]
Cross-linking agents for proteins include:[citation needed]
Chaotropic agents include:[citation needed]
Agents that breakdisulfide bonds by reduction include:[citation needed]
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.
Acidic nucleic acid denaturants include:
Basic nucleic acid denaturants include:
Other nucleic acid denaturants include:
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