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Denaturation (biochemistry)

(Redirected from DNA denaturation)

In biochemistry, denaturation is a process in which proteins or nucleic acids lose folded structure present in their native state due to various factors, including 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.[3] 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.[4][5] Denatured proteins can exhibit a wide range of characteristics, from conformational change and loss of solubility or dissociation of cofactors to aggregation due to the exposure of hydrophobic groups. The loss of solubility as a result of denaturation is called coagulation.[6] Denatured proteins lose their 3D structure, and therefore, cannot function.

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

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 of bioactivity.

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: An enzyme loses its ability to alter or speed up a chemical reaction when it is denaturized.[2]

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.

Common examples

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(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.[7] 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.[8]

Protein denaturation

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See also: Equilibrium unfolding

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

 
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:
  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

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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.[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 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.[11]

How denaturation occurs at levels of protein structure

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See also: Protein structure

Loss of function

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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,[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 as dual-polarization interferometry, CD, QCM-D and MP-SPR.

Loss of activity due to heavy metals and metalloids

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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 of metalloid.[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.

Reversibility and irreversibility

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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.[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 the DNA that codes for the protein, the so-called "Anfinsen's thermodynamic 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]

Protein denaturation due to pH

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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]

Nucleic acid denaturation

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Main article: Nucleic acid thermodynamics

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

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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.[19] 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.[19]

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.[20] 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.[21]

Recent thermodynamic studies have inferred that the lifetime of a singular denaturation bubble ranges from 1 microsecond to 1 millisecond.[22] This information is based on established timescales of DNA replication and transcription.[22] Currently,[when?] biophysical and biochemical research studies are being performed to more fully elucidate the thermodynamic details of the denaturation bubble.[22]

Denaturation due to chemical agents

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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.[23] 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.[24] 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.[23] 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.[25]

Chemical denaturation as an alternative

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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.[24][26][27] 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.[23] 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.[23]

Denaturation due to air

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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.[28] 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.[28] Antiparellel strands in DNA double helices are non-covalently bound by hydrogen bonding between base pairs;[29] nitrogen and oxygen therefore maintain the potential to weaken the integrity of DNA when exposed to air.[30] As a result, DNA strands exposed to air require less force to separate and exemplify lower melting temperatures.[30]

Applications

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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

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Protein denaturants

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Acids

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Acidic protein denaturants include:

Bases

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Bases work similarly to acids in denaturation. They include:

Solvents

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

Cross-linking reagents

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

Chaotropic agents

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Chaotropic agents include:[citation needed]

Disulfide bond reducers

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

Chemically reactive agents

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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

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Nucleic acid denaturants

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Chemical

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Acidic nucleic acid denaturants include:

Basic nucleic acid denaturants include:

  • NaOH

Other nucleic acid denaturants include:

Physical

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See also

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References

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  1. ^ Alan D. MacNaught; Andrew R. Wilkinson, eds. (1997). Compendium of Chemical Terminology: IUPAC Recommendations (the "Gold Book"). Blackwell Science. ISBN 978-0865426849.
  2. ^ Vert, Michel (2012). "Terminology for biorelated polymers and applications (IUPAC Recommendations 2012)" (PDF). Pure and Applied Chemistry. 84 (2): 377–410. doi:10.1351/PAC-REC-10-12-04. S2CID 98107080. Archived (PDF) from the original on 2013-09-27.
  3. ^ Mosby's Medical Dictionary (8th ed.). Elsevier. 2009. Retrieved 1 October 2013.
  4. ^ Samson, Andre L.; Ho, Bosco; Au, Amanda E.; Schoenwaelder, Simone M.; Smyth, Mark J.; Bottomley, Stephen P.; Kleifeld, Oded; Medcalf, Robert L. (2016-11-01). "Physicochemical properties that control protein aggregation also determine whether a protein is retained or released from necrotic cells". Open Biology. 6 (11): 160098. doi:10.1098/rsob.160098. ISSN 2046-2441. PMC 5133435. PMID 27810968.
  5. ^ Samson, Andre L.; Knaupp, Anja S.; Sashindranath, Maithili; Borg, Rachael J.; Au, Amanda E.-L.; Cops, Elisa J.; Saunders, Helen M.; Cody, Stephen H.; McLean, Catriona A. (2012-10-25). "Nucleocytoplasmic coagulation: an injury-induced aggregation event that disulfide crosslinks proteins and facilitates their removal by plasmin". Cell Reports. 2 (4): 889–901. doi:10.1016/j.celrep.2012.08.026. ISSN 2211-1247. PMID 23041318.
  6. ^ "2.5: Denaturation of proteins". Chemistry LibreTexts. 2019-07-15. Retrieved 2022-04-25.
  7. ^ Mine, Yoshinori; Noutomi, Tatsushi; Haga, Noriyuki (1990). "Thermally induced changes in egg white proteins". Journal of Agricultural and Food Chemistry. 38 (12): 2122–2125. Bibcode:1990JAFC...38.2122M. doi:10.1021/jf00102a004.
  8. ^ "Ceviche: the new sushi," The Times.
  9. ^ a b c Bondos, Sarah (2014). "Protein folding". Access Science. doi:10.1036/1097-8542.801070.
  10. ^ "Denaturation". Science in Context. 2006-04-03.
  11. ^ Dyson, H. Jane; Wright, Peter E. (2005-03-01). "Intrinsically unstructured proteins and their functions". Nature Reviews Molecular Cell Biology. 6 (3): 197–208. doi:10.1038/nrm1589. ISSN 1471-0072. PMID 15738986. S2CID 18068406.
  12. ^ Charles Tanford (1968), "Protein denaturation" (PDF), Advances in Protein Chemistry, 23: 121–282, doi:10.1016/S0065-3233(08)60401-5, ISBN 9780120342235, PMID 4882248, archived (PDF) from the original on 2005-11-10
  13. ^ Biology Online Dictionary (2 December 2020), Denaturation Definition and Examples
  14. ^ a b c d e f Tamás, Markus J.; Sharma, Sandeep K.; Ibstedt, Sebastian; Jacobson, Therese; Christen, Philipp (2014-03-04). "Heavy Metals and Metalloids As a Cause for Protein Misfolding and Aggregation". Biomolecules. 4 (1): 252–267. doi:10.3390/biom4010252. PMC 4030994. PMID 24970215.
  15. ^ Campbell, N. A.; Reece, J.B.; Meyers, N.; Urry, L. A.; Cain, M.L.; Wasserman, S.A.; Minorsky, P.V.; Jackson, R.B. (2009), Biology (8th, Australian version ed.), Sydney: Pearson Education Australia
  16. ^ Anfinsen CB. (1973), "Principles that govern the folding of protein chains", Science, 181 (4096): 223–30, Bibcode:1973Sci...181..223A, doi:10.1126/science.181.4096.223, PMID 4124164, S2CID 10151090
  17. ^ Wetlaufer, D.B. (1988). "Reversible and irreversible denaturation of proteins in chromatographic systems". Makromolekulare Chemie. Macromolecular Symposia. 17 (1): 17–28. doi:10.1002/masy.19880170104. ISSN 0258-0322.
  18. ^ a b Konermann, Lars (2012-05-15). "Protein Unfolding and Denaturants". Encyclopedia of Life Sciences. Chichester, UK: John Wiley & Sons, Ltd. doi:10.1002/9780470015902.a0003004.pub2. ISBN 978-0470016176. {{cite book}}: |journal= ignored (help)
  19. ^ a b Sicard, François; Destainville, Nicolas; Manghi, Manoel (21 January 2015). "DNA denaturation bubbles: Free-energy landscape and nucleation/closure rates". The Journal of Chemical Physics. 142 (3): 034903. arXiv:1405.3867. Bibcode:2015JChPh.142c4903S. doi:10.1063/1.4905668. PMID 25612729. S2CID 13967558.
  20. ^ Lieu, Simon. "The Poland-Scheraga Model." (2015): 0-5. Massachusetts Institute of Technology, 14 May 2015. Web. 25 Oct. 2016.
  21. ^ Richard, C., and A. J. Guttmann. "Poland–Scheraga Models and the DNA Denaturation Transition." Journal of Statistical Physics 115.3/4 (2004): 925-47. Web.
  22. ^ a b c Altan-Bonnet, Grégoire; Libchaber, Albert; Krichevsky, Oleg (1 April 2003). "Bubble Dynamics in Double-Stranded DNA". Physical Review Letters. 90 (13): 138101. Bibcode:2003PhRvL..90m8101A. doi:10.1103/physrevlett.90.138101. PMID 12689326. S2CID 1427570.
  23. ^ a b c d Wang, X (2014). "Characterization of denaturation and renaturation of DNA for DNA hybridization". Environmental Health and Toxicology. 29: e2014007. doi:10.5620/eht.2014.29.e2014007. PMC 4168728. PMID 25234413.
  24. ^ a b Marmur, J (1961). "Denaturation of deoxyribonucleic acid by formamide". Biochimica et Biophysica Acta. 51 (1): 91013–7. doi:10.1016/0006-3002(61)91013-7. PMID 13767022.
  25. ^ "Denaturing Polyacrylamide Gel Electrophoresis of DNA & RNA". Electrophoresis. National Diagnostics. 15 August 2011. Retrieved 13 October 2016.
  26. ^ Tinoco, I; Bustamante, C; Maestre, M (1980). "The Optical Activity of Nucleic Acids and their Aggregates". Annual Review of Biophysics and Bioengineering. 9 (1): 107–141. doi:10.1146/annurev.bb.09.060180.000543. PMID 6156638.
  27. ^ Fernandes, M (2002). "Calculation of hydrodynamic properties of small nucleic acids from their atomic structure". Nucleic Acids Research. 30 (8): 1782–8. doi:10.1093/nar/30.8.1782. PMC 113193. PMID 11937632.
  28. ^ a b Mathers, T. L.; Schoeffler, G.; McGlynn, S. P. (July 1985). "The effects of selected gases upon ethanol: hydrogen bond breaking by O and N". Canadian Journal of Chemistry. 63 (7): 1864–1869. doi:10.1139/v85-309.
  29. ^ Cox, David L. Nelson, Michael M. (2008). Lehninger principles of biochemistry (5th ed.). New York: W.H. Freeman. ISBN 9780716771081.{{cite book}}: CS1 maint: multiple names: authors list (link)
  30. ^ a b Mathers, T. L.; Schoeffler, G.; McGlynn, S. P. (1982). "Hydrogen-bond breaking by O/sub 2/ and N/sub 2/. II. Melting curves of DNA" (PDF). doi:10.2172/5693881. OSTI 5693881. Archived (PDF) from the original on 2018-07-24. {{cite journal}}: Cite journal requires |journal= (help)
  31. ^ López-Alonso JP, Bruix M, Font J, Ribó M, Vilanova M, Jiménez MA, Santoro J, González C, Laurents DV (2010), "NMR spectroscopy reveals that RNase A is chiefly denatured in 40% acetic acid: implications for oligomer formation by 3D domain swapping", J. Am. Chem. Soc., 132 (5): 1621–30, doi:10.1021/ja9081638, PMID 20085318
  32. ^ Jaremko, M.; Jaremko Ł; Kim HY; Cho MK; Schwieters CD; Giller K; Becker S; Zweckstetter M. (April 2013). "Cold denaturation of a protein dimer monitored at atomic resolution". Nat. Chem. Biol. 9 (4): 264–70. doi:10.1038/nchembio.1181. PMC 5521822. PMID 23396077.
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