View source for Protein folding

{{short description|Change of a linear protein chain to a 3D structure}}
<!-- courtesy note per [[WP:RSECT]]: [[Misfoldings]] and several others redirect here -->

[[Image:Protein folding.png|thumb|right|360px|Protein before and after folding]]
[[File:Protein structure.png|right|360px|thumb|Results of protein folding]]
'''Protein folding''' is the [[physical process]] by which a [[protein]], after [[Protein biosynthesis|synthesis]] by a [[ribosome]] as a linear chain of [[Amino acid|amino acids]], changes from an unstable [[random coil]] into a more ordered [[protein tertiary structure|three-dimensional structure]]. This structure permits the protein to become biologically functional or active.<ref name=Alberts>{{cite book | last1 = Alberts | first1 = Bruce | first2 = Alexander | last2 = Johnson | first3 = Julian | last3 = Lewis | first4 = Martin | last4 = Raff | first5 = Keith | last5 = Roberts | first6 = Peter | last6 = Walters | name-list-style = vanc | title = Molecular Biology of the Cell; Fourth Edition | publisher = Garland Science| year = 2002 | location = New York and London | chapter = The Shape and Structure of Proteins | chapter-url = https://www.ncbi.nlm.nih.gov/books/NBK26830/ | isbn = 978-0-8153-3218-3 | author-link1 = Bruce Alberts}}</ref>

The folding of many proteins begins even during the translation of the polypeptide chain. The amino acids interact with each other to produce a well-defined three-dimensional structure, known as the protein's [[native state]]. This structure is determined by the amino-acid sequence or [[primary structure]].<ref name="Anfinsen">{{cite journal | vauthors = Anfinsen CB | title = The formation and stabilization of protein structure | journal = The Biochemical Journal | volume = 128 | issue = 4 | pages = 737–49 | date = July 1972 | pmid = 4565129 | pmc = 1173893 | doi = 10.1042/bj1280737 }}</ref>

The correct three-dimensional structure is essential to function, although some parts of functional proteins [[Intrinsically unstructured proteins|may remain unfolded]],<ref>{{cite book | first1 = Jeremy M. | last1 = Berg | first2 = John L. | last2 = Tymoczko | first3 = Lubert | last3 = Stryer | author-link3 = Lubert Stryer | name-list-style = vanc | title = Biochemistry | publisher = W. H. Freeman | location = San Francisco | year = 2002 | isbn = 978-0-7167-4684-3 | chapter = 3. Protein Structure and Function | chapter-url = https://www.ncbi.nlm.nih.gov/books?cmd=Search&doptcmdl=GenBookHL&term=stryer%5Bbook%5D%20AND%20215168%5Buid%5D&rid=stryer.chapter.280}}</ref> indicating that [[protein dynamics]] are important. Failure to fold into a native structure generally produces inactive proteins, but in some instances, misfolded proteins have modified or toxic functionality. Several [[neurodegenerative]] and other [[disease]]s are believed to result from the accumulation of [[amyloid]] [[fibrils]] formed by misfolded proteins, the infectious varieties of which are known as [[Prion|prions]].<ref name="Selkoe:03">{{cite journal | vauthors = Selkoe DJ | title = Folding proteins in fatal ways | journal = Nature | volume = 426 | issue = 6968 | pages = 900–4 | date = December 2003 | pmid = 14685251 | doi = 10.1038/nature02264 | bibcode = 2003Natur.426..900S | s2cid = 6451881 }}</ref> Many [[Protein allergy|allergies]] are caused by the incorrect folding of some proteins because the [[immune system]] does not produce the [[antibodies]] for certain protein structures.<ref>{{cite book | last1 = Alberts | first1 = Bruce | first2 = Dennis | last2 = Bray | first3 = Karen | last3 = Hopkin | first4 = Alexander | last4 = Johnson | first5 = Julian | last5 = Lewis | first6 = Martin | last6 = Raff | first7 = Keith | last7 = Roberts | first8 = Peter | last8 = Walter | name-list-style = vanc | title = Essential cell biology | date = 2010 | publisher = Garland Science | location = New York, NY | isbn = 978-0-8153-4454-4 | pages = 120–70 | edition = Third | chapter = Protein Structure and Function}}</ref>

[[Denaturation (biochemistry)|Denaturation]] of proteins is a process of transition from a folded to an [[Random coil|unfolded state]]. It happens in [[cooking]], [[burn]]s, [[proteinopathy|proteinopathies]], and other contexts.  Residual structure present, if any, in the supposedly unfolded state may form a folding initiation site and guide the subsequent folding reactions.<ref>{{cite journal | vauthors = Yagi-Utsumi M, Chandak MS, Yanaka S, Hiranyakorn M, Nakamura T, Kato K, Kuwajima K| title = Residual Structure of Unfolded Ubiquitin as Revealed by Hydrogen/Deuterium-Exchange 2D NMR | journal = Biophysical Journal | volume = 119 | issue = 10 | pages = 2029–38 | date = November 2020 | pmid = 33142107 | doi = 10.1016/j.bpj.2020.10.003 | pmc = 7732725 | bibcode = 2020BpJ...119.2029Y }}</ref>

The duration of the folding process varies dramatically depending on the protein of interest. When studied [[in vitro|outside the cell]], the slowest folding proteins require many minutes or hours to fold, primarily due to [[Proline#Cis-trans isomerization|proline isomerization]], and must pass through a number of intermediate states, like checkpoints, before the process is complete.<ref>{{cite journal | vauthors = Kim PS, Baldwin RL | title = Intermediates in the folding reactions of small proteins | journal = Annual Review of Biochemistry | volume = 59 | pages = 631–60 | year = 1990 | pmid = 2197986 | doi = 10.1146/annurev.bi.59.070190.003215 }}</ref> On the other hand, very small single-[[protein domain|domain]] proteins with lengths of up to a hundred amino acids typically fold in a single step.<ref>{{cite journal | vauthors = Jackson SE | title = How do small single-domain proteins fold? | journal = Folding & Design | volume = 3 | issue = 4 | pages = R81-91 | year = 1998 | pmid = 9710577 | doi = 10.1016/S1359-0278(98)00033-9 | doi-access =  }}</ref> Time scales of milliseconds are the norm, and the fastest known protein folding reactions are complete within a few microseconds.<ref>{{cite journal | vauthors = Kubelka J, Hofrichter J, Eaton WA | title = The protein folding 'speed limit' | journal = Current Opinion in Structural Biology | volume = 14 | issue = 1 | pages = 76–88 | date = February 2004 | pmid = 15102453 | doi = 10.1016/j.sbi.2004.01.013 | url = https://zenodo.org/record/1259347 }} </ref> The folding time scale of a protein depends on its size, [[contact order]], and [[circuit topology]].<ref>{{Cite journal | url=https://pubs.rsc.org/en/content/articlelanding/2021/cp/d1cp03390e | doi=10.1039/D1CP03390E | title=Topological principles of protein folding | year=2021 | last1=Scalvini | first1=Barbara | last2=Sheikhhassani | first2=Vahid | last3=Mashaghi | first3=Alireza | journal=Physical Chemistry Chemical Physics | volume=23 | issue=37 | pages=21316–21328 | pmid=34545868 | bibcode=2021PCCP...2321316S | hdl=1887/3277889 | s2cid=237583577 | hdl-access=free }}</ref>

Understanding and simulating the protein folding process has been an important challenge for [[computational biology]] since the late 1960s.

== Process of protein folding ==
=== Primary structure ===
{{Main|Protein primary structure}}

The primary structure of a protein, its linear amino-acid sequence, determines its native conformation.<ref name="Anfinsen1">{{cite journal | vauthors = Anfinsen CB | title = Principles that govern the folding of protein chains | journal = Science | volume = 181 | issue = 4096 | pages = 223–30 | date = July 1973 | pmid = 4124164 | doi = 10.1126/science.181.4096.223 | bibcode = 1973Sci...181..223A }}</ref> The specific amino acid residues and their position in the polypeptide chain are the determining factors for which portions of the protein fold closely together and form its three-dimensional conformation. The amino acid composition is not as important as the sequence.<ref name="Voet_2016">{{cite book | title = Principles of Biochemistry | first1 = Donald | last1 = Voet | first2 = Judith G. | last2 = Voet | first3 = Charlotte W. | last3 = Pratt | name-list-style = vanc | publisher = Wiley | year = 2016 | edition = Fifth | isbn = 978-1-118-91840-1 }}</ref> The essential fact of folding, however, remains that the amino acid sequence of each protein contains the information that specifies both the native structure and the pathway to attain that state. This is not to say that nearly identical amino acid sequences always fold similarly.<ref>{{cite journal | vauthors = Alexander PA, He Y, Chen Y, Orban J, Bryan PN | title = The design and characterization of two proteins with 88% sequence identity but different structure and function | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 104 | issue = 29 | pages = 11963–8 | date = July 2007 | pmid = 17609385 | pmc = 1906725 | doi = 10.1073/pnas.0700922104 | bibcode = 2007PNAS..10411963A | doi-access = free }}</ref> Conformations differ based on environmental factors as well; similar proteins fold differently based on where they are found.

=== Secondary structure ===
{{Main|Protein secondary structure}}
[[File:Alpha helix.png|thumb|332x332px|The [[alpha helix]] spiral formation]]
[[File:BetaPleatedSheetProtein.png|left|thumb|150x150px|An anti-parallel [[beta pleated sheet]] displaying hydrogen bonding within the backbone]]

Formation of a [[Protein secondary structure|secondary structure]] is the first step in the folding process that a protein takes to assume its native structure. Characteristic of secondary structure are the structures known as [[alpha helix|alpha helices]] and [[beta sheet]]s that fold rapidly because they are stabilized by [[Intramolecular force|intramolecular]] [[hydrogen bond]]s, as was first characterized by [[Linus Pauling]]. Formation of intramolecular hydrogen bonds provides another important contribution to protein stability.<ref name="Rose">{{cite journal | vauthors = Rose GD, Fleming PJ, Banavar JR, Maritan A | title = A backbone-based theory of protein folding | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 103 | issue = 45 | pages = 16623–33 | date = November 2006 | pmid = 17075053 | pmc = 1636505 | doi = 10.1073/pnas.0606843103 | bibcode = 2006PNAS..10316623R | citeseerx = 10.1.1.630.5487 | doi-access = free }}</ref> α-helices are formed by hydrogen bonding of the [[Backbone chain|backbone]] to form a spiral shape (refer to figure on the right).<ref name="Voet_2016" /> The β pleated sheet is a structure that forms with the backbone bending over itself to form the hydrogen bonds (as displayed in the figure to the left). The hydrogen bonds are between the amide hydrogen and carbonyl oxygen of the [[peptide bond]]. There exists anti-parallel β pleated sheets and parallel β pleated sheets where the stability of the hydrogen bonds is stronger in the anti-parallel β sheet as it hydrogen bonds with the ideal 180 degree angle compared to the slanted hydrogen bonds formed by parallel sheets.<ref name="Voet_2016" />

=== Tertiary structure ===
{{Main|Protein tertiary structure}}

The α-Helices and β-Sheets are commonly amphipathic, meaning they have a hydrophilic and a hydrophobic portion. This ability helps in forming tertiary structure of a protein in which folding occurs so that the hydrophilic sides are facing the aqueous environment surrounding the protein and the hydrophobic sides are facing the hydrophobic core of the protein.<ref name="Fersht_1999" /> Secondary structure hierarchically gives way to tertiary structure formation. Once the protein's tertiary structure is formed and stabilized by the hydrophobic interactions, there may also be [[covalent bond]]ing in the form of [[disulfide bond|disulfide bridges]] formed between two [[cysteine]] residues. These non-covalent and covalent contacts take a specific [[circuit topology|topological]] arrangement in a native structure of a protein. Tertiary structure of a protein involves a single polypeptide chain; however, additional interactions of folded polypeptide chains give rise to quaternary structure formation.<ref>{{cite web | url = http://www.nature.com/scitable/topicpage/protein-structure-14122136 | title = Protein Structure | publisher = Nature Education | access-date = 2016-11-26 | work = Scitable }}</ref>

=== Quaternary structure ===
{{Main|Protein quaternary structure}}

Tertiary structure may give way to the formation of quaternary structure in some proteins, which usually involves the "assembly" or "coassembly" of subunits that have already folded; in other words, multiple polypeptide chains could interact to form a fully functional quaternary protein.<ref name="Voet_2016" />

=== Driving forces of protein folding ===
[[File:225 Peptide Bond-01.jpg|thumb|263x263px|All forms of protein structure summarized]]

Folding is a [[spontaneous process]] that is mainly guided by hydrophobic interactions, formation of intramolecular [[hydrogen bond]]s, [[van der Waals forces]], and it is opposed by [[conformational entropy]].<ref>{{cite book | first1 = Charlotte | last1 = Pratt | first2 = Kathleen | last2 = Cornely | name-list-style = vanc | chapter-url = http://www.wiley.com/college/pratt/0471393878/instructor/review/thermodynamics/7_relationship.html | chapter = Thermodynamics | title = Essential Biochemistry | publisher = Wiley | access-date = 2016-11-26 | date = 2004 | isbn = 978-0-471-39387-0 | url-access = registration | url = https://archive.org/details/essentialbiochem00char }}</ref> The folding time scale of an isolated protein depends on its size, [[contact order]], and [[circuit topology]]. Inside cells, the process of folding often begins [[translation (genetics)|co-translationally]], so that the [[N-terminus]] of the protein begins to fold while the [[C-terminus|C-terminal]] portion of the protein is still being [[protein biosynthesis|synthesized]] by the [[ribosome]]; however, a protein molecule may fold spontaneously during or after [[Protein biosynthesis|biosynthesis]].<ref>{{cite journal | vauthors = Zhang G, Ignatova Z | title = Folding at the birth of the nascent chain: coordinating translation with co-translational folding | journal = Current Opinion in Structural Biology | volume = 21 | issue = 1 | pages = 25–31 | date = February 2011 | pmid = 21111607 | doi = 10.1016/j.sbi.2010.10.008 }}</ref> While these [[macromolecule]]s may be regarded as "[[Self-assembly|folding themselves]]", the process also depends on the [[solvent]] ([[water]] or [[lipid bilayer]]),<ref>{{cite journal | vauthors = van den Berg B, Wain R, Dobson CM, Ellis RJ | title = Macromolecular crowding perturbs protein refolding kinetics: implications for folding inside the cell | journal = The EMBO Journal | volume = 19 | issue = 15 | pages = 3870–5 | date = August 2000 | pmid = 10921869 | pmc = 306593 | doi = 10.1093/emboj/19.15.3870 }}</ref> the concentration of [[Salt (chemistry)|salts]], the [[pH]], the [[temperature]], the possible presence of cofactors and of molecular [[Chaperone (protein)|chaperone]]s.

Proteins will have limitations on their folding abilities by the restricted bending angles or conformations that are possible. These allowable angles of protein folding are described with a two-dimensional plot known as the [[Ramachandran plot]], depicted with psi and phi angles of allowable rotation.<ref>{{cite web | url = http://www.proteinstructures.com/Structure/Structure/Ramachandran-plot.html | title = Torsion Angles and the Ramachnadran Plot in Protein Structures | first =  Salam | last = Al-Karadaghi | name-list-style = vanc | work = www.proteinstructures.com | access-date = 2016-11-26}}</ref>

==== Hydrophobic effect ====
[[Image:Protein folding schematic.png|thumb|181x181px|[[Hydrophobic collapse]]. In the compact fold (to the right), the hydrophobic amino acids (shown as black spheres) collapse toward the center to become shielded from aqueous environment.|left]]

Protein folding must be thermodynamically favorable within a cell in order for it to be a spontaneous reaction. Since it is known that protein folding is a spontaneous reaction, then it must assume a negative [[Gibbs free energy]] value. Gibbs free energy in protein folding is directly related to [[enthalpy]] and [[entropy]].<ref name="Voet_2016" /> For a negative delta G to arise and for protein folding to become thermodynamically favorable, then either enthalpy, entropy, or both terms must be favorable.

[[File:Molecular Dynamics Simulation of the Hydrophobic Solvation of Argon.webm|thumb|Entropy is decreased as the water molecules become more orderly near the hydrophobic solute.|262x262px]]

Minimizing the number of hydrophobic side-chains exposed to water is an important driving force behind the folding process.<ref name="Pace">{{cite journal | vauthors = Pace CN, Shirley BA, McNutt M, Gajiwala K | title = Forces contributing to the conformational stability of proteins | journal = FASEB Journal | volume = 10 | issue = 1 | pages = 75–83 | date = January 1996 | pmid = 8566551 | doi = 10.1096/fasebj.10.1.8566551 | doi-access = free | s2cid = 20021399 }}</ref> The hydrophobic effect is the phenomenon in which the hydrophobic chains of a protein collapse into the core of the protein (away from the hydrophilic environment).<ref name="Voet_2016" /> In an aqueous environment, the water molecules tend to aggregate around the hydrophobic regions or side chains of the protein, creating water shells of ordered water molecules.<ref>{{cite journal | vauthors = Cui D, Ou S, Patel S | title = Protein-spanning water networks and implications for prediction of protein–protein interactions mediated through hydrophobic effects | journal = Proteins | volume = 82 | issue = 12 | pages = 3312–26 | date = December 2014 | pmid = 25204743 | doi = 10.1002/prot.24683 | s2cid = 27113763 }}</ref> An ordering of water molecules around a hydrophobic region increases order in a system and therefore contributes a negative change in entropy (less entropy in the system). The water molecules are fixed in these water cages which drives the [[hydrophobic collapse]], or the inward folding of the hydrophobic groups. The hydrophobic collapse introduces entropy back to the system via the breaking of the water cages which frees the ordered water molecules.<ref name="Voet_2016" /> The multitude of hydrophobic groups interacting within the core of the globular folded protein contributes a significant amount to protein stability after folding, because of the vastly accumulated van der Waals forces (specifically [[London dispersion force|London Dispersion forces]]).<ref name="Voet_2016" /> The [[hydrophobic effect]] exists as a driving force in thermodynamics only if there is the presence of an aqueous medium with an [[amphiphilic]] molecule containing a large hydrophobic region.<ref>{{cite journal | vauthors = Tanford C | title = The hydrophobic effect and the organization of living matter | journal = Science | volume = 200 | issue = 4345 | pages = 1012–8 | date = June 1978 | pmid = 653353 | doi = 10.1126/science.653353 | bibcode = 1978Sci...200.1012T }}</ref> The strength of hydrogen bonds depends on their environment; thus, H-bonds enveloped in a hydrophobic core contribute more than H-bonds exposed to the aqueous environment to the stability of the native state.<ref name="Deechongkit">{{cite journal | vauthors = Deechongkit S, Nguyen H, Powers ET, Dawson PE, Gruebele M, Kelly JW | title = Context-dependent contributions of backbone hydrogen bonding to beta-sheet folding energetics | journal = Nature | volume = 430 | issue = 6995 | pages = 101–5 | date = July 2004 | pmid = 15229605 | doi = 10.1038/nature02611 | bibcode = 2004Natur.430..101D | s2cid = 4315026 }}</ref>

In proteins with globular folds, hydrophobic amino acids tend to be interspersed along the primary sequence, rather than randomly distributed or clustered together.<ref>{{cite journal | vauthors = Irbäck A, Sandelin E | title = On hydrophobicity correlations in protein chains | journal = Biophysical Journal | volume = 79 | issue = 5 | pages = 2252–8 | date = November 2000 | pmid = 11053106 | pmc = 1301114 | doi = 10.1016/S0006-3495(00)76472-1 | arxiv = cond-mat/0010390 | bibcode = 2000BpJ....79.2252I }}</ref><ref>{{cite journal | vauthors = Irbäck A, Peterson C, Potthast F | title = Evidence for nonrandom hydrophobicity structures in protein chains | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 93 | issue = 18 | pages = 9533–8 | date = September 1996 | pmid = 8790365 | pmc = 38463 | doi = 10.1073/pnas.93.18.9533 | arxiv = chem-ph/9512004 | bibcode = 1996PNAS...93.9533I | doi-access = free }}</ref> However, proteins that have recently been born [[De novo gene birth|de novo]], which tend to be [[intrinsically disordered proteins|intrinsically disordered]],<ref>{{cite journal | vauthors = Wilson BA, Foy SG, Neme R, Masel J | title = De Novo Gene Birth | journal = Nature Ecology & Evolution | volume = 1 | issue = 6 | pages = 0146–146 | date = June 2017 | pmid = 28642936 | pmc = 5476217 | doi = 10.1038/s41559-017-0146 | bibcode = 2017NatEE...1..146W }}</ref><ref>{{cite journal | vauthors = Willis S, Masel J | title = Gene Birth Contributes to Structural Disorder Encoded by Overlapping Genes | journal = Genetics | volume = 210 | issue = 1 | pages = 303–313 | date = September 2018 | pmid = 30026186 | pmc = 6116962 | doi = 10.1534/genetics.118.301249 }}</ref> show the opposite pattern of hydrophobic amino acid clustering along the primary sequence.<ref>{{cite journal | vauthors = Foy SG, Wilson BA, Bertram J, Cordes MH, Masel J | title = A Shift in Aggregation Avoidance Strategy Marks a Long-Term Direction to Protein Evolution | journal = Genetics | volume = 211 | issue = 4 | pages = 1345–1355 | date = April 2019 | pmid = 30692195 | pmc = 6456324 | doi = 10.1534/genetics.118.301719 }}</ref>

==== Chaperones ====
[[File:PDB 1gme EBI.jpg|thumb|Example of a small eukaryotic [[heat shock protein]]]]

[[Chaperone (protein)|Molecular chaperones]] are a class of proteins that aid in the correct folding of other proteins ''[[in vivo]]''. Chaperones exist in all cellular compartments and interact with the polypeptide chain in order to allow the native three-dimensional conformation of the protein to form; however, chaperones themselves are not included in the final structure of the protein they are assisting in.<ref name="Dobson_2003" /> Chaperones may assist in folding even when the nascent polypeptide is being synthesized by the ribosome.<ref name="Hartl_1996" /> Molecular chaperones operate by binding to stabilize an otherwise unstable structure of a protein in its folding pathway, but chaperones do not contain the necessary information to know the correct native structure of the protein they are aiding; rather, chaperones work by preventing incorrect folding conformations.<ref name="Hartl_1996">{{cite journal | vauthors = Hartl FU | title = Molecular chaperones in cellular protein folding | journal = Nature | volume = 381 | issue = 6583 | pages = 571–9 | date = June 1996 | pmid = 8637592 | doi = 10.1038/381571a0 | bibcode = 1996Natur.381..571H | s2cid = 4347271 }}</ref> In this way, chaperones do not actually increase the rate of individual steps involved in the folding pathway toward the native structure; instead, they work by reducing possible unwanted aggregations of the polypeptide chain that might otherwise slow down the search for the proper intermediate and they provide a more efficient pathway for the polypeptide chain to assume the correct conformations.<ref name="Dobson_2003" /> Chaperones are not to be confused with folding [[Catalysis|catalyst]] proteins, which catalyze chemical reactions responsible for slow steps in folding pathways. Examples of folding catalysts are protein [[disulfide isomerase]]s and [[peptidyl-prolyl isomerase]]s that may be involved in formation of [[disulfide bond]]s or interconversion between cis and trans stereoisomers of peptide group.<ref name="Hartl_1996" /> Chaperones are shown to be critical in the process of protein folding ''in vivo'' because they provide the protein with the aid needed to assume its proper alignments and conformations efficiently enough to become "biologically relevant".<ref name="Hartl_2011">{{cite journal | vauthors = Hartl FU, Bracher A, Hayer-Hartl M | title = Molecular chaperones in protein folding and proteostasis | journal = Nature | volume = 475 | issue = 7356 | pages = 324–32 | date = July 2011 | pmid = 21776078 | doi = 10.1038/nature10317 | s2cid = 4337671 }}</ref> This means that the polypeptide chain could theoretically fold into its native structure without the aid of chaperones, as demonstrated by protein folding experiments conducted ''[[in vitro]]'';<ref name="Hartl_2011" /> however, this process proves to be too inefficient or too slow to exist in biological systems; therefore, chaperones are necessary for protein folding ''in vivo.'' Along with its role in aiding native structure formation, chaperones are shown to be involved in various roles such as protein transport, degradation, and even allow [[Denaturation (biochemistry)|denatured proteins]] exposed to certain external denaturant factors an opportunity to refold into their correct native structures.<ref>{{cite journal | vauthors = Kim YE, Hipp MS, Bracher A, Hayer-Hartl M, Hartl FU | title = Molecular chaperone functions in protein folding and proteostasis | journal = Annual Review of Biochemistry | volume = 82 | pages = 323–55 | year = 2013 | pmid = 23746257 | doi = 10.1146/annurev-biochem-060208-092442 }}</ref>

A fully denatured protein lacks both tertiary and secondary structure, and exists as a so-called [[random coil]]. Under certain conditions some proteins can refold; however, in many cases, denaturation is irreversible.<ref name="Shortle">{{cite journal | vauthors = Shortle D | title = The denatured state (the other half of the folding equation) and its role in protein stability | journal = FASEB Journal | volume = 10 | issue = 1 | pages = 27–34 | date = January 1996 | pmid = 8566543 | doi = 10.1096/fasebj.10.1.8566543 | doi-access = free | s2cid = 24066207 }}</ref> Cells sometimes protect their proteins against the denaturing influence of heat with [[enzyme]]s known as [[heat shock protein]]s (a type of chaperone), which assist other proteins both in folding and in remaining folded.  [[Heat shock protein]]s have been found in all species examined, from [[bacteria]] to humans, suggesting that they evolved very early and have an important function.  Some proteins never fold in cells at all except with the assistance of chaperones which either isolate individual proteins so that their folding is not interrupted by interactions with other proteins or help to unfold misfolded proteins, allowing them to refold into the correct native structure.<ref name="Lee_2005">{{cite journal | vauthors = Lee S, Tsai FT | title = Molecular chaperones in protein quality control | journal = Journal of Biochemistry and Molecular Biology | volume = 38 | issue = 3 | pages = 259–65 | year = 2005 | pmid = 15943899 | doi = 10.5483/BMBRep.2005.38.3.259 | doi-access = free }}</ref> This function is crucial to prevent the risk of [[precipitation (chemistry)|precipitation]] into [[insoluble]] amorphous aggregates. The external factors involved in protein denaturation or disruption of the native state include temperature, external fields (electric, magnetic),<ref name="ojeda">{{cite journal | vauthors = Ojeda-May P, Garcia ME | title = Electric field-driven disruption of a native beta-sheet protein conformation and generation of a helix-structure | journal = Biophysical Journal | volume = 99 | issue = 2 | pages = 595–9 | date = July 2010 | pmid = 20643079 | pmc = 2905109 | doi = 10.1016/j.bpj.2010.04.040 | bibcode = 2010BpJ....99..595O }}</ref> molecular crowding,<ref name="berg">{{cite journal | vauthors = van den Berg B, Ellis RJ, Dobson CM | title = Effects of macromolecular crowding on protein folding and aggregation | journal = The EMBO Journal | volume = 18 | issue = 24 | pages = 6927–33 | date = December 1999 | pmid = 10601015 | pmc = 1171756 | doi = 10.1093/emboj/18.24.6927 }}</ref> and even the limitation of space (i.e. confinement), which can have a big influence on the folding of proteins.<ref>{{cite journal | vauthors = Ellis RJ | title = Molecular chaperones: assisting assembly in addition to folding | journal = Trends in Biochemical Sciences | volume = 31 | issue = 7 | pages = 395–401 | date = July 2006 | pmid = 16716593 | doi = 10.1016/j.tibs.2006.05.001 }}</ref> High concentrations of [[solutes]], extremes of [[pH]], mechanical forces, and the presence of chemical denaturants can contribute to protein denaturation, as well. These individual factors are categorized together as stresses. Chaperones are shown to exist in increasing concentrations during times of cellular stress and help the proper folding of emerging proteins as well as denatured or misfolded ones.<ref name="Dobson_2003" />

Under some conditions proteins will not fold into their biochemically functional forms. Temperatures above or below the range that cells tend to live in will cause [[Thermostability|thermally unstable]] proteins to unfold or denature (this is why boiling makes an [[Egg white#Denaturation|egg white]] turn opaque). Protein thermal stability is far from constant, however; for example, [[hyperthermophiles|hyperthermophilic bacteria]] have been found that grow at temperatures as high as 122&nbsp;°C,<ref>{{cite journal | vauthors = Takai K, Nakamura K, Toki T, Tsunogai U, Miyazaki M, Miyazaki J, Hirayama H, Nakagawa S, Nunoura T, Horikoshi K | title = Cell proliferation at 122 degrees C and isotopically heavy CH4 production by a hyperthermophilic methanogen under high-pressure cultivation | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 105 | issue = 31 | pages = 10949–54 | date = August 2008 | pmid = 18664583 | pmc = 2490668 | doi = 10.1073/pnas.0712334105 | bibcode = 2008PNAS..10510949T | doi-access = free }}</ref> which of course requires that their full complement of vital proteins and protein assemblies be stable at that temperature or above.

The bacterium ''[[E. coli]]'' is the host for [[bacteriophage T4]], and the phage encoded gp31 protein ({{UniProt|P17313}}) appears to be structurally and functionally homologous to ''E. coli'' chaperone protein [[GroES]] and able to substitute for it in the assembly of bacteriophage T4 [[virus]] particles during infection.<ref name = Marusich1998>{{cite journal |last1=Marusich |first1=EI |last2=Kurochkina |first2=LP |last3=Mesyanzhinov |first3=VV |title=Chaperones in bacteriophage T4 assembly |journal=Biochemistry. Biokhimiia |date=April 1998 |volume=63 |issue=4 |pages=399–406 |pmid=9556522 |url=http://www.protein.bio.msu.ru/biokhimiya/contents/v63/full/63040473.html }}</ref> Like GroES, gp31 forms a stable complex with [[GroEL]] chaperonin that is absolutely necessary for the folding and assembly in vivo of the bacteriophage T4 major capsid protein gp23.<ref name = Marusich1998/>

=== Fold switching ===
Some proteins have multiple native structures, and change their fold based on some external factors. For example, the KaiB protein [[KaiB#Circadian outputs and KaiB fold switching|switches fold throughout the day]], acting as a clock for cyanobacteria. It has been estimated that around 0.5&ndash;4% of PDB ([[Protein Data Bank]]) proteins switch folds.<ref>{{cite journal |last1=Porter |first1=Lauren L. |last2=Looger |first2=Loren L. |title=Extant fold-switching proteins are widespread |journal=Proceedings of the National Academy of Sciences |date=5 June 2018 |volume=115 |issue=23 |pages=5968–5973 |doi=10.1073/pnas.1800168115 |pmid=29784778 |pmc=6003340 |bibcode=2018PNAS..115.5968P |doi-access=free}}</ref>

== Protein misfolding and neurodegenerative disease ==
{{Main|Proteopathy}}
A protein is considered to be [[protein misfolding|misfolded]] if it cannot achieve its normal native state. This can be due to mutations in the amino acid sequence or a disruption of the normal folding process by external factors.<ref name="Chaudhuri_2006"/> The misfolded protein typically contains [[Beta sheet|β-sheets]] that are organized in a supramolecular arrangement known as a cross-β structure. These β-sheet-rich assemblies are very stable, very insoluble, and generally resistant to proteolysis.<ref name="Soto_2006">{{cite journal | vauthors = Soto C, Estrada L, Castilla J | title = Amyloids, prions and the inherent infectious nature of misfolded protein aggregates | journal = Trends in Biochemical Sciences | volume = 31 | issue = 3 | pages = 150–5 | date = March 2006 | pmid = 16473510 | doi = 10.1016/j.tibs.2006.01.002 }}</ref> The structural stability of these fibrillar assemblies is caused by extensive interactions between the protein monomers, formed by backbone hydrogen bonds between their β-strands.<ref name="Soto_2006" /> The misfolding of proteins can trigger the further misfolding and accumulation of other proteins into aggregates or oligomers. The increased levels of aggregated proteins in the cell leads to formation of [[amyloid]]-like structures which can cause degenerative disorders and cell death.<ref name="Chaudhuri_2006">{{cite journal | vauthors = Chaudhuri TK, Paul S | title = Protein-misfolding diseases and chaperone-based therapeutic approaches | journal = The FEBS Journal | volume = 273 | issue = 7 | pages = 1331–49 | date = April 2006 | pmid = 16689923 | doi = 10.1111/j.1742-4658.2006.05181.x | s2cid = 23370420 | doi-access = free }}</ref> The amyloids are fibrillary structures that contain intermolecular hydrogen bonds which are highly insoluble and made from converted protein aggregates.<ref name="Chaudhuri_2006" /> Therefore, the proteasome pathway may not be efficient enough to degrade the misfolded proteins prior to aggregation. Misfolded proteins can interact with one another and form structured aggregates and gain toxicity through intermolecular interactions.<ref name="Chaudhuri_2006" />

Aggregated proteins are associated with [[prion]]-related illnesses such as [[Creutzfeldt–Jakob disease]], [[bovine spongiform encephalopathy]] (mad cow disease), amyloid-related illnesses such as [[Alzheimer's disease]] and [[familial amyloid cardiomyopathy]] or [[Familial amyloid polyneuropathy|polyneuropathy]],<ref name="pmid12560553">{{cite journal | vauthors = Hammarström P, Wiseman RL, Powers ET, Kelly JW | title = Prevention of transthyretin amyloid disease by changing protein misfolding energetics | journal = Science | volume = 299 | issue = 5607 | pages = 713–6 | date = January 2003 | pmid = 12560553 | doi = 10.1126/science.1079589 | bibcode = 2003Sci...299..713H | s2cid = 30829998 }}</ref> as well as intracellular aggregation diseases such as [[Huntington's]] and [[Parkinson's disease]].<ref name="Selkoe:03" /><ref name="ChitiDobson">{{cite journal | vauthors = Chiti F, Dobson CM | title = Protein misfolding, functional amyloid, and human disease | journal = Annual Review of Biochemistry | volume = 75 | pages = 333–66 | year = 2006 | pmid = 16756495 | doi = 10.1146/annurev.biochem.75.101304.123901 | s2cid = 23797549 }}</ref> These age onset degenerative diseases are associated with the aggregation of misfolded proteins into insoluble, extracellular aggregates and/or intracellular inclusions including cross-β [[amyloid]] [[fibril]]s. It is not completely clear whether the aggregates are the cause or merely a reflection of the loss of protein homeostasis, the balance between synthesis, folding, aggregation and protein turnover. Recently the [[European Medicines Agency]] approved the use of [[Tafamidis]] or Vyndaqel (a kinetic stabilizer of tetrameric transthyretin) for the treatment of transthyretin amyloid diseases. This suggests that the process of amyloid fibril formation (and not the fibrils themselves) causes the degeneration of post-mitotic tissue in human amyloid diseases.<ref name="pmid16359163">{{cite journal | vauthors = Johnson SM, Wiseman RL, Sekijima Y, Green NS, Adamski-Werner SL, Kelly JW | title = Native state kinetic stabilization as a strategy to ameliorate protein misfolding diseases: a focus on the transthyretin amyloidoses | journal = Accounts of Chemical Research | volume = 38 | issue = 12 | pages = 911–21 | date = December 2005 | pmid = 16359163 | doi = 10.1021/ar020073i }}</ref> Misfolding and excessive degradation instead of folding and function leads to a number of [[proteopathy]] diseases such as [[antitrypsin]]-associated [[emphysema]], [[cystic fibrosis]] and the [[lysosomal storage diseases]], where loss of function is the origin of the disorder. While protein replacement therapy has historically been used to correct the latter disorders, an emerging approach is to use [[pharmaceutical chaperones]] to fold mutated proteins to render them functional.

== Experimental techniques for studying protein folding ==
While inferences about protein folding can be made through [[Phi value analysis|mutation studies]], typically, experimental techniques for studying protein folding rely on the [[Equilibrium unfolding|gradual unfolding]] or folding of proteins and observing conformational changes using standard non-crystallographic techniques.

===X-ray crystallography===
[[File:X ray diffraction.png|thumb|Steps of [[X-ray crystallography]]|321x321px]]
[[X-ray crystallography]] is one of the more efficient and important methods for attempting to decipher the three dimensional configuration of a folded protein.<ref name="Cowtan_2001">{{cite encyclopedia | url = http://people.bu.edu/mfk/restricted566/phaseproblem.pdf | title = Phase Problem in X-ray Crystallography, and Its Solution | last = Cowtan | first = Kevin | name-list-style = vanc | date = 2001 | encyclopedia = Encyclopedia of Life Sciences |publisher=Macmillan Publishers Ltd, Nature Publishing Group|access-date=November 3, 2016}}</ref> To be able to conduct X-ray crystallography, the protein under investigation must be located inside a crystal lattice. To place a protein inside a crystal lattice, one must have a suitable solvent for crystallization, obtain a pure protein at supersaturated levels in solution, and precipitate the crystals in solution.<ref>{{cite book | url=https://books.google.com/books?id=Jobr7svN0IIC&pg=PR5 |title = Principles of Protein X-Ray Crystallography | last = Drenth | first = Jan | name-list-style = vanc | date = 2007-04-05 | publisher = Springer Science & Business Media | isbn = 978-0-387-33746-3 }}</ref> Once a protein is crystallized, X-ray beams can be concentrated through the crystal lattice which would diffract the beams or shoot them outwards in various directions. These exiting beams are correlated to the specific three-dimensional configuration of the protein enclosed within. The X-rays specifically interact with the electron clouds surrounding the individual atoms within the protein crystal lattice and produce a discernible diffraction pattern.<ref name="Fersht_1999">{{cite book |url=https://books.google.com/books?id=QdpZz_ahA5UC&pg=PR20 |title=Structure and Mechanism in Protein Science: A Guide to Enzyme Catalysis and Protein Folding | last = Fersht | first = Alan | name-list-style = vanc | date = 1999 | publisher = Macmillan | isbn = 978-0-7167-3268-6 }}</ref> Only by relating the electron density clouds with the amplitude of the X-rays can this pattern be read and lead to assumptions of the phases or phase angles involved that complicate this method.<ref>{{cite journal |doi=10.1107/S0907444903017815 |title=The phase problem |journal=Acta Crystallographica Section D |volume=59 |issue=11 |pages=1881–90 |year=2003 |last1=Taylor |first1=Garry | name-list-style = vanc |pmid=14573942 |doi-access=free |bibcode=2003AcCrD..59.1881T }}</ref> Without the relation established through a mathematical basis known as [[Fourier transform]], the "[[phase problem]]" would render predicting the diffraction patterns very difficult.<ref name="Fersht_1999" /> Emerging methods like [[multiple isomorphous replacement]] use the presence of a heavy metal ion to diffract the X-rays into a more predictable manner, reducing the number of variables involved and resolving the phase problem.<ref name="Cowtan_2001" />

===Fluorescence spectroscopy===
[[Fluorescence spectroscopy]] is a highly sensitive method for studying the folding state of proteins. Three amino acids, phenylalanine (Phe), tyrosine (Tyr) and tryptophan (Trp), have intrinsic fluorescence properties, but only Tyr and Trp are used experimentally because their [[quantum yield]]s are high enough to give good fluorescence signals. Both Trp and Tyr are excited by a wavelength of 280&nbsp;nm, whereas only Trp is excited by a wavelength of 295&nbsp;nm. Because of their aromatic character, Trp and Tyr residues are often found fully or partially buried in the hydrophobic core of proteins, at the interface between two protein domains, or at the interface between subunits of oligomeric proteins. In this apolar environment, they have high quantum yields and therefore high fluorescence intensities. Upon disruption of the protein's tertiary or quaternary structure, these side chains become more exposed to the hydrophilic environment of the solvent, and their quantum yields decrease, leading to low fluorescence intensities. For Trp residues, the wavelength of their maximal fluorescence emission also depend on their environment.

Fluorescence spectroscopy can be used to characterize the [[equilibrium unfolding]] of proteins by measuring the variation in the intensity of fluorescence emission or in the wavelength of maximal emission as functions of a denaturant value.<ref name="pmid=26607240">{{cite journal | vauthors = Bedouelle H | title = Principles and equations for measuring and interpreting protein stability: From monomer to tetramer | journal = Biochimie | volume = 121 | pages = 29–37 | date = February 2016 | pmid = 26607240 | doi = 10.1016/j.biochi.2015.11.013 }}</ref><ref>{{cite journal | vauthors = Monsellier E, Bedouelle H | title = Quantitative measurement of protein stability from unfolding equilibria monitored with the fluorescence maximum wavelength | journal = Protein Engineering, Design & Selection | volume = 18 | issue = 9 | pages = 445–56 | date = September 2005 | pmid = 16087653 | doi = 10.1093/protein/gzi046 | doi-access = free }}</ref> The denaturant can be a chemical molecule (urea, guanidinium hydrochloride), temperature, pH, pressure, etc. The equilibrium between the different but discrete protein states, i.e. native state, intermediate states, unfolded state, depends on the denaturant value; therefore, the global fluorescence signal of their equilibrium mixture also depends on this value. One thus obtains a profile relating the global protein signal to the denaturant value. The profile of equilibrium unfolding may enable one to detect and identify intermediates of unfolding.<ref>{{cite journal | vauthors = Park YC, Bedouelle H | title = Dimeric tyrosyl-tRNA synthetase from Bacillus stearothermophilus unfolds through a monomeric intermediate. A quantitative analysis under equilibrium conditions | journal = The Journal of Biological Chemistry | volume = 273 | issue = 29 | pages = 18052–9 | date = July 1998 | pmid = 9660761 | doi = 10.1074/jbc.273.29.18052 | doi-access = free }}</ref><ref>{{cite journal | vauthors = Ould-Abeih MB, Petit-Topin I, Zidane N, Baron B, Bedouelle H | title = Multiple folding states and disorder of ribosomal protein SA, a membrane receptor for laminin, anticarcinogens, and pathogens | journal = Biochemistry | volume = 51 | issue = 24 | pages = 4807–21 | date = June 2012 | pmid = 22640394 | doi = 10.1021/bi300335r }}</ref> General equations have been developed by Hugues Bedouelle to obtain the thermodynamic parameters that characterize the unfolding equilibria for homomeric or heteromeric proteins, up to trimers and potentially tetramers, from such profiles.<ref name="pmid=26607240"/> Fluorescence spectroscopy can be combined with fast-mixing devices such as [[stopped flow]], to measure protein folding kinetics,<ref>{{cite journal | vauthors = Royer CA | title = Probing protein folding and conformational transitions with fluorescence | journal = Chemical Reviews | volume = 106 | issue = 5 | pages = 1769–84 | date = May 2006 | pmid = 16683754 | doi = 10.1021/cr0404390 }}</ref> generate a [[chevron plot]] and derive a [[Phi value analysis]].

===Circular dichroism===
{{Main|Circular dichroism}}

[[Circular dichroism]] is one of the most general and basic tools to study protein folding. Circular dichroism spectroscopy measures the absorption of [[circular polarization|circularly polarized light]]. In proteins, structures such as [[alpha helix|alpha helices]] and [[beta sheets]] are chiral, and thus absorb such light. The absorption of this light acts as a marker of the degree of foldedness of the protein ensemble. This technique has been used to measure [[equilibrium unfolding]] of the protein by measuring the change in this absorption as a function of denaturant concentration or [[temperature]]. A denaturant melt measures the [[Thermodynamic free energy|free energy]] of unfolding as well as the protein's m value, or denaturant dependence. A temperature melt measures the [[Denaturation midpoint|denaturation temperature]] (Tm) of the protein.<ref name="pmid=26607240" /> As for fluorescence spectroscopy, circular-dichroism spectroscopy can be combined with fast-mixing devices such as [[stopped flow]] to measure protein folding [[Chemical kinetics|kinetics]] and to generate [[chevron plot]]s.

===Vibrational circular dichroism of proteins===
The more recent developments of [[vibrational circular dichroism]] (VCD) techniques for proteins, currently involving [[Fourier transform]] (FT) instruments, provide powerful means for determining protein conformations in solution even for very large protein molecules. Such VCD studies of proteins can be combined with [[X-ray diffraction]] data for protein crystals, [[FT-IR]] data for protein solutions in heavy water (D<sub>2</sub>O), or [[Quantum chemistry|quantum computations]].

===Protein nuclear magnetic resonance spectroscopy===
{{Main|Protein NMR}}

Protein [[nuclear magnetic resonance]] (NMR) is able to collect protein structural data by inducing a magnet field through samples of concentrated protein. In NMR, depending on the chemical environment, certain nuclei will absorb specific radio-frequencies.<ref name=":1">{{cite journal | vauthors = Wüthrich K | title = Protein structure determination in solution by NMR spectroscopy | journal = The Journal of Biological Chemistry | volume = 265 | issue = 36 | pages = 22059–62 | date = December 1990 | doi = 10.1016/S0021-9258(18)45665-7 | pmid = 2266107 | doi-access = free }}</ref><ref name=":0">{{cite journal | vauthors = Zhuravleva A, Korzhnev DM | title = Protein folding by NMR | journal = Progress in Nuclear Magnetic Resonance Spectroscopy | volume = 100 | pages = 52–77 | date = May 2017 | pmid = 28552172 | doi = 10.1016/j.pnmrs.2016.10.002 | bibcode = 2017PNMRS.100...52Z | url = http://www.sciencedirect.com/science/article/pii/S0079656516300280 | url-access = subscription }}</ref> Because protein structural changes operate on a time scale from ns to ms, NMR is especially equipped to study intermediate structures in timescales of ps to s.<ref name=":5">{{cite book |doi=10.1016/b978-0-12-411636-8.00006-7 |chapter=Protein Functional Dynamics in Multiple Timescales as Studied by NMR Spectroscopy |title=Dynamics of Proteins and Nucleic Acids |series=Advances in Protein Chemistry and Structural Biology |year=2013 |last1=Ortega |first1=Gabriel |last2=Pons |first2=Miquel |last3=Millet |first3=Oscar |volume=92 |pages=219–251 |pmid=23954103 |isbn=978-0-12-411636-8 }}</ref> Some of the main techniques for studying proteins structure and non-folding protein structural changes include [[Two-dimensional nuclear magnetic resonance spectroscopy|COSY]], [[Two-dimensional nuclear magnetic resonance spectroscopy|TOCSY]], [[Heteronuclear single quantum coherence spectroscopy|HSQC]], [[Relaxation (NMR)|time relaxation]] (T1 & T2), and [[Nuclear Overhauser effect|NOE]].<ref name=":1" /> NOE is especially useful because magnetization transfers can be observed between spatially proximal hydrogens are observed.<ref name=":1" /> Different NMR experiments have varying degrees of timescale sensitivity that are appropriate for different protein structural changes. NOE can pick up bond vibrations or side chain rotations, however, NOE is too sensitive to pick up protein folding because it occurs at larger timescale.<ref name=":5" />[[File:Protein Structural changes timescale matched with NMR experiments.png|thumb|350x350px|Timescale of protein structural changes matched with NMR experiments. For protein folding, CPMG Relaxation Dispersion (CPMG RD) and chemical exchange saturation transfer (CEST) collect data in the appropriate timescale.]]

Because protein folding takes place in about 50 to 3000 s<sup>−1</sup> CPMG Relaxation dispersion and [[Magnetization transfer|chemical exchange saturation transfer]] have become some of the primary techniques for NMR analysis of folding.<ref name=":0" /> In addition, both techniques are used to uncover excited intermediate states in the protein folding landscape.<ref name=":4">{{cite journal | vauthors = Vallurupalli P, Bouvignies G, Kay LE | title = Studying "invisible" excited protein states in slow exchange with a major state conformation | journal = Journal of the American Chemical Society | volume = 134 | issue = 19 | pages = 8148–61 | date = May 2012 | pmid = 22554188 | doi = 10.1021/ja3001419 | bibcode = 2012JAChS.134.8148V }}</ref> To do this, CPMG Relaxation dispersion takes advantage of the [[spin echo]] phenomenon. This technique exposes the target nuclei to a 90 pulse followed by one or more 180 pulses.<ref name=":2">{{cite journal | vauthors = Neudecker P, Lundström P, Kay LE | title = Relaxation dispersion NMR spectroscopy as a tool for detailed studies of protein folding | journal = Biophysical Journal | volume = 96 | issue = 6 | pages = 2045–54 | date = March 2009 | pmid = 19289032 | pmc = 2717354 | doi = 10.1016/j.bpj.2008.12.3907 | bibcode = 2009BpJ....96.2045N }}</ref> As the nuclei refocus, a broad distribution indicates the target nuclei is involved in an intermediate excited state. By looking at Relaxation dispersion plots the data collect information on the thermodynamics and kinetics between the excited and ground.<ref name=":2" /><ref name=":4" /> Saturation Transfer measures changes in signal from the ground state as excited states become perturbed. It uses weak radio frequency irradiation to saturate the excited state of a particular nuclei which transfers its saturation to the ground state.<ref name=":0" /> This signal is amplified by decreasing the magnetization (and the signal) of the ground state.<ref name=":0" /><ref name=":4" />

The main limitations in NMR is that its resolution decreases with proteins that are larger than 25 kDa and is not as detailed as [[X-ray crystallography]].<ref name=":0" /> Additionally, protein NMR analysis is quite difficult and can propose multiple solutions from the same NMR spectrum.<ref name=":1" />

In a study focused on the folding of an [[amyotrophic lateral sclerosis]] involved protein [[SOD1]], excited intermediates were studied with relaxation dispersion and Saturation transfer.<ref name=":3">{{cite journal | vauthors = Sekhar A, Rumfeldt JA, Broom HR, Doyle CM, Sobering RE, Meiering EM, Kay LE | title = Probing the free energy landscapes of ALS disease mutants of SOD1 by NMR spectroscopy | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 113 | issue = 45 | pages = E6939–E6945 | date = November 2016 | pmid = 27791136 | pmc = 5111666 | doi = 10.1073/pnas.1611418113 | bibcode = 2016PNAS..113E6939S | doi-access = free }}</ref> SOD1 had been previously tied to many disease causing mutants which were assumed to be involved in protein aggregation, however the mechanism was still unknown. By using Relaxation Dispersion and Saturation Transfer experiments many excited intermediate states were uncovered misfolding in the SOD1 mutants.<ref name=":3" />

=== Dual-polarization interferometry ===
{{Main|Dual-polarization interferometry}}
[[Dual polarisation interferometry]] is a surface-based technique for measuring the optical properties of molecular layers. When used to characterize protein folding, it measures the [[protein conformation|conformation]] by determining the overall size of a monolayer of the protein and its density in real time at sub-Angstrom resolution,<ref name="CrossFreeman2008">{{cite book |doi=10.1002/9780470061565.hbb055 |chapter=Dual Polarization Interferometry: A Real-Time Optical Technique for Measuring (Bio)molecular Orientation, Structure and Function at the Solid/Liquid Interface |title=Handbook of Biosensors and Biochips |year=2008 |last1=Cross |first1=Graham H. |last2=Freeman |first2=Neville J. |last3=Swann |first3=Marcus J. | name-list-style = vanc |isbn=978-0-470-01905-4 }}</ref> although real-time measurement of the kinetics of protein folding are limited to processes that occur slower than ~10&nbsp;Hz. Similar to [[circular dichroism]], the stimulus for folding can be a denaturant or [[temperature]].

=== Studies of folding with high time resolution ===
The study of protein folding has been greatly advanced in recent years by the development of fast, time-resolved techniques. Experimenters rapidly trigger the folding of a sample of unfolded protein and observe the resulting [[protein dynamics|dynamics]]. Fast techniques in use include ultrafast mixing of solutions, photochemical methods, and [[Temperature jump|laser temperature jump spectroscopy]]. Among the many scientists who have contributed to the development of these techniques are Heinrich Roder, Terry Oas, [[Harry Gray (chemist)|Harry Gray]], [[Martin Gruebele]], Brian Dyer, William Eaton, [[Sheena Radford]], [[Chris Dobson]], [[Alan Fersht]], [[Bengt Nölting]] and Lars Konermann.

=== Proteolysis ===
[[Proteolysis]] is routinely used to probe the fraction unfolded under a wide range of solution conditions (e.g. [[fast parallel proteolysis (FASTpp)]].<ref name="Minde">{{cite journal | vauthors = Minde DP, Maurice MM, Rüdiger SG | title = Determining biophysical protein stability in lysates by a fast proteolysis assay, FASTpp | journal = PLOS ONE | volume = 7 | issue = 10 | article-number = e46147 | year = 2012 | pmid = 23056252 | pmc = 3463568 | doi = 10.1371/journal.pone.0046147 | bibcode = 2012PLoSO...746147M | doi-access = free }}</ref><ref name="Park">{{cite journal | vauthors = Park C, Marqusee S | title = Pulse proteolysis: a simple method for quantitative determination of protein stability and ligand binding | journal = Nature Methods | volume = 2 | issue = 3 | pages = 207–12 | date = March 2005 | pmid = 15782190 | doi = 10.1038/nmeth740 | s2cid = 21364478 }}</ref>

=== Single-molecule force spectroscopy ===
Single molecule techniques such as optical tweezers and AFM have been used to understand protein folding mechanisms of isolated proteins as well as proteins with chaperones.<ref name="pmid24001118">{{cite journal | vauthors = Mashaghi A, Kramer G, Lamb DC, Mayer MP, Tans SJ | title = Chaperone action at the single-molecule level | journal = Chemical Reviews | volume = 114 | issue = 1 | pages = 660–76 | date = January 2014 | pmid = 24001118 | doi = 10.1021/cr400326k }}</ref> [[Optical tweezers]] have been used to stretch single protein molecules from their C- and N-termini and unfold them to allow study of the subsequent refolding.<ref>{{cite journal | vauthors = Jagannathan B, Marqusee S | title = Protein folding and unfolding under force | journal = Biopolymers | volume = 99 | issue = 11 | pages = 860–9 | date = November 2013 | pmid = 23784721 | pmc = 4065244 | doi = 10.1002/bip.22321 }}</ref> The technique allows one to measure folding rates at single-molecule level; for example, optical tweezers have been recently applied to study folding and unfolding of proteins involved in blood coagulation. [[von Willebrand factor]] (vWF) is a protein with an essential role in blood clot formation process. It discovered – using single molecule optical tweezers measurement – that calcium-bound vWF acts as a shear force sensor in the blood. Shear force leads to unfolding of the A2 domain of vWF, whose refolding rate is dramatically enhanced in the presence of calcium.<ref>{{cite journal | vauthors = Jakobi AJ, Mashaghi A, Tans SJ, Huizinga EG | title = Calcium modulates force sensing by the von Willebrand factor A2 domain | journal = Nature Communications | volume = 2 | article-number = 385 | date = July 2011 | pmid = 21750539 | pmc = 3144584 | doi = 10.1038/ncomms1385 | bibcode = 2011NatCo...2..385J }}</ref> Recently, it was also shown that the simple src [[SH3 domain]] accesses multiple unfolding pathways under force.<ref>{{cite journal | vauthors = Jagannathan B, Elms PJ, Bustamante C, Marqusee S | title = Direct observation of a force-induced switch in the anisotropic mechanical unfolding pathway of a protein | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 109 | issue = 44 | pages = 17820–5 | date = October 2012 | pmid = 22949695 | pmc = 3497811 | doi = 10.1073/pnas.1201800109 | bibcode = 2012PNAS..10917820J | doi-access = free }}</ref>

=== Biotin painting ===
Biotin painting enables condition-specific cellular snapshots of (un)folded proteins. Biotin 'painting' shows a bias towards predicted [[Intrinsically disordered proteins]].<ref name="Biotinylation by proximity labelling favours unfolded proteins">{{cite journal | vauthors = Minde DP, Ramakrishna M, Lilley KS | title = Biotinylation by proximity labelling favours unfolded proteins | journal = bioRxiv | year = 2018 | doi = 10.1101/274761 | doi-access = free | url = https://www.biorxiv.org/content/biorxiv/early/2018/09/13/274761.full.pdf }}</ref>

== Computational studies of protein folding{{anchor|Computational_methods_for_studying_protein_folding}} ==
Computational studies of protein folding includes three main aspects related to the prediction of protein stability, kinetics, and structure. A 2013 review summarizes the available computational methods for protein folding. 
<ref>{{cite journal | vauthors = Compiani M, Capriotti E | title = Computational and theoretical methods for protein folding | journal = Biochemistry | volume = 52 | issue = 48 | pages = 8601–24 | date = December 2013 | pmid = 24187909 | doi = 10.1021/bi4001529 }}</ref>

=== Levinthal's paradox ===
In 1969, Cyrus Levinthal noted that, because of the very large number of degrees of freedom in an unfolded polypeptide chain, the molecule has an astronomical number of possible conformations. An estimate of 3<sup>300</sup> or 10<sup>143</sup> was made in one of his papers.<ref>{{Cite web|url=https://en.wikibooks.org/wiki/Structural_Biochemistry/Proteins/Protein_Folding#The_Levinthal_Paradox|title=Structural Biochemistry/Proteins/Protein Folding - Wikibooks, open books for an open world|website=en.wikibooks.org|access-date=2016-11-05}}</ref> [[Levinthal's paradox]] is a thought experiment based on the observation that if a protein were folded by sequential sampling of all possible conformations, it would take an astronomical amount of time to do so, even if the conformations were sampled at a rapid rate (on the [[nanosecond]] or [[picosecond]] scale).<ref>{{cite journal | last = Levinthal | first = Cyrus | name-list-style = vanc | year = 1968 | title = Are there pathways for protein folding? | url = http://www.biochem.wisc.edu/courses/biochem704/Reading/Levinthal1968.pdf | journal = Journal de Chimie Physique et de Physico-Chimie Biologique | volume = 65 | pages = 44–45 | archive-url = https://web.archive.org/web/20090902211239/http://www.biochem.wisc.edu/courses/biochem704/Reading/Levinthal1968.pdf | archive-date = 2009-09-02 | doi = 10.1051/jcp/1968650044 | bibcode = 1968JCP....65...44L }}</ref> Based upon the observation that proteins fold much faster than this, Levinthal then proposed that a random conformational search does not occur, and the protein must, therefore, fold through a series of meta-stable [[Reaction intermediate|intermediate states]].

=== Energy landscape of protein folding ===
[[File:Folding funnel schematic.svg|thumb|286x286px|The energy funnel by which an unfolded polypeptide chain assumes its native structure]]
The [[Configuration space (physics)|configuration space]] of a protein during folding can be visualized as an [[energy landscape]]. According to Joseph Bryngelson and [[Peter Wolynes]], proteins follow the ''principle of minimal frustration'', meaning that naturally evolved proteins have optimized their folding energy landscapes,<ref name="bryngelson">{{cite journal | vauthors = Bryngelson JD, Onuchic JN, Socci ND, Wolynes PG | title = Funnels, pathways, and the energy landscape of protein folding: a synthesis | journal = Proteins | volume = 21 | issue = 3 | pages = 167–95 | date = March 1995 | pmid = 7784423 | doi = 10.1002/prot.340210302 | arxiv = chem-ph/9411008 | s2cid = 13838095 }}</ref> and that nature has chosen amino acid sequences so that the folded state of the protein is sufficiently stable. In addition, the acquisition of the folded state had to become a sufficiently fast process. Even though nature has reduced the level of ''frustration'' in proteins, some degree of it remains up to now as can be observed in the presence of local minima in the energy landscape of proteins.

A consequence of these evolutionarily selected sequences is that proteins are generally thought to have globally "funneled energy landscapes" (a term coined by [[José Onuchic]])<ref>{{cite journal | vauthors = Leopold PE, Montal M, Onuchic JN | author-link3 = José Onuchic | title = Protein folding funnels: a kinetic approach to the sequence-structure relationship | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 89 | issue = 18 | pages = 8721–5 | date = September 1992 | pmid = 1528885 | pmc = 49992 | doi = 10.1073/pnas.89.18.8721 | bibcode = 1992PNAS...89.8721L | doi-access = free }}</ref> that are largely directed toward the native state. This "[[folding funnel]]" landscape allows the protein to fold to the native state through any of a large number of pathways and intermediates, rather than being restricted to a single mechanism. The theory is supported by both [[lattice protein|computational simulations of model proteins]] and experimental studies,<ref name="bryngelson" /> and it has been used to improve methods for [[protein structure prediction]] and [[protein design|design]].<ref name="bryngelson" /> The description of protein folding by the leveling free-energy landscape is also consistent with the 2nd law of thermodynamics.<ref>{{cite journal |doi=10.1016/j.physa.2008.12.004 |title=Protein folding as an evolutionary process |journal=Physica A: Statistical Mechanics and Its Applications | volume = 388 | issue = 6 | pages = 851–62 | year = 2009 | last1 = Sharma | first1 = Vivek | last2 = Kaila | first2 = Ville R.I. | last3 = Annila | first3 = Arto | name-list-style = vanc  | bibcode = 2009PhyA..388..851S }}</ref> Physically, thinking of landscapes in terms of visualizable potential or total energy surfaces simply with maxima, saddle points, minima, and funnels, rather like geographic landscapes, is perhaps a little misleading. The relevant description is really a high-dimensional phase space in which manifolds might take a variety of more complicated topological forms.<ref name="Robson_2008">{{cite book |doi=10.1016/S0079-6603(08)00405-4 |pmid=19121702 |chapter=Protein Folding Revisited |title=Molecular Biology of Protein Folding, Part B |volume=84 |pages=161–202 |series=Progress in Molecular Biology and Translational Science |year=2008 |last1=Robson |first1=Barry |last2=Vaithilingam |first2=Andy | name-list-style = vanc |isbn=978-0-12-374595-8 }}</ref>

The unfolded polypeptide chain begins at the top of the funnel where it may assume the largest number of unfolded variations and is in its highest energy state. Energy landscapes such as these indicate that there are a large number of initial possibilities, but only a single native state is possible; however, it does not reveal the numerous folding pathways that are possible. A different molecule of the same exact protein may be able to follow marginally different folding pathways, seeking different lower energy intermediates, as long as the same native structure is reached.<ref name="Dill_2012">{{cite journal | vauthors = Dill KA, MacCallum JL | title = The protein-folding problem, 50 years on | journal = Science | volume = 338 | issue = 6110 | pages = 1042–6 | date = November 2012 | pmid = 23180855 | doi = 10.1126/science.1219021 | bibcode = 2012Sci...338.1042D | s2cid = 5756068 }}</ref> Different pathways may have different frequencies of utilization depending on the thermodynamic favorability of each pathway. This means that if one pathway is found to be more thermodynamically favorable than another, it is likely to be used more frequently in the pursuit of the native structure.<ref name="Dill_2012" /> As the protein begins to fold and assume its various conformations, it always seeks a more thermodynamically favorable structure than before and thus continues through the energy funnel. Formation of secondary structures is a strong indication of increased stability within the protein, and only one combination of secondary structures assumed by the polypeptide backbone will have the lowest energy and therefore be present in the native state of the protein.<ref name="Dill_2012" /> Among the first structures to form once the polypeptide begins to fold are alpha helices and beta turns, where alpha helices can form in as little as 100 nanoseconds and beta turns in 1 microsecond.<ref name="Dobson_2003">{{cite journal | vauthors = Dobson CM | title = Protein folding and misfolding | journal = Nature | volume = 426 | issue = 6968 | pages = 884–90 | date = December 2003 | pmid = 14685248 | doi = 10.1038/nature02261 | bibcode = 2003Natur.426..884D | s2cid = 1036192 }}</ref>

There exists a saddle point in the energy funnel landscape where the [[transition state]] for a particular protein is found.<ref name="Dobson_2003" /> The transition state in the energy funnel diagram is the conformation that must be assumed by every molecule of that protein if the protein wishes to finally assume the native structure. No protein may assume the native structure without first passing through the transition state.<ref name="Dobson_2003" /> The transition state can be referred to as a variant or premature form of the native state rather than just another intermediary step.<ref name="Fersht_2000">{{cite journal | vauthors = Fersht AR | title = Transition-state structure as a unifying basis in protein-folding mechanisms: contact order, chain topology, stability, and the extended nucleus mechanism | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 97 | issue = 4 | pages = 1525–9 | date = February 2000 | pmid = 10677494 | pmc = 26468 | doi = 10.1073/pnas.97.4.1525 | bibcode = 2000PNAS...97.1525F | doi-access = free }}</ref> The folding of the transition state is shown to be rate-determining, and even though it exists in a higher energy state than the native fold, it greatly resembles the native structure. Within the transition state, there exists a nucleus around which the protein is able to fold, formed by a process referred to as "nucleation condensation" where the structure begins to collapse onto the nucleus.<ref name="Fersht_2000" />

=== Modeling of protein folding ===
 [[File:ACBP MSM from Folding@home.tiff|right|thumb|350px|Folding@home uses [[Markov state model]]s, like the one diagrammed here, to model the possible shapes and folding pathways a protein can take as it condenses from its initial randomly coiled state (left) into its native 3D structure (right).]]

''[[wiktionary:de novo|De novo]]'' or ''[[ab initio]]'' techniques for computational [[protein structure prediction]] can be used for simulating various aspects of protein folding. [[Molecular dynamics]] (MD) was used in simulations of protein folding and dynamics [[in silico]].<ref name="Rizzuti">{{cite journal | vauthors = Rizzuti B, Daggett V | title = Using simulations to provide the framework for experimental protein folding studies | journal = Archives of Biochemistry and Biophysics | volume = 531 | issue = 1–2 | pages = 128–35 | date = March 2013 | pmid = 23266569 | pmc = 4084838 | doi = 10.1016/j.abb.2012.12.015 }}</ref> First equilibrium folding simulations were done using implicit solvent model and [[umbrella sampling]].<ref>{{cite journal | vauthors = Schaefer M, Bartels C, Karplus M | title = Solution conformations and thermodynamics of structured peptides: molecular dynamics simulation with an implicit solvation model | journal = Journal of Molecular Biology | volume = 284 | issue = 3 | pages = 835–48 | date = December 1998 | pmid = 9826519 | doi = 10.1006/jmbi.1998.2172 }}</ref> Because of computational cost, ab initio MD folding simulations with explicit water are limited to peptides and small proteins.<ref>{{cite web | url = http://www.cs.ucl.ac.uk/staff/d.jones/t42morph.html | title = Fragment-based Protein Folding Simulations | first = David | last = Jones | name-list-style = vanc | publisher = University College London }}</ref><ref>{{cite web | url = http://www.biomolecular-modeling.com/Abalone/Protein-folding.html | title = Protein folding | format = by Molecular Dynamics }}</ref> MD simulations of larger proteins remain restricted to dynamics of the experimental structure or its high-temperature unfolding. Long-time folding processes (beyond about 1 millisecond), like folding of larger proteins (>150 residues)  can be accessed using [[Coarse-grained modeling|coarse-grained models]].<ref>{{cite journal | vauthors = Kmiecik S, Gront D, Kolinski M, Wieteska L, Dawid AE, Kolinski A | title = Coarse-Grained Protein Models and Their Applications | journal = Chemical Reviews | volume = 116 | issue = 14 | pages = 7898–936 | date = July 2016 | pmid = 27333362 | doi = 10.1021/acs.chemrev.6b00163 | doi-access = free }}</ref><ref name="Kmiecik">{{cite journal | vauthors = Kmiecik S, Kolinski A | title = Characterization of protein-folding pathways by reduced-space modeling | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 104 | issue = 30 | pages = 12330–5 | date = July 2007 | pmid = 17636132 | pmc = 1941469 | doi = 10.1073/pnas.0702265104 | bibcode = 2007PNAS..10412330K | doi-access = free }}</ref><ref name="teritfix">{{cite journal | vauthors = Adhikari AN, Freed KF, Sosnick TR | title = De novo prediction of protein folding pathways and structure using the principle of sequential stabilization | journal = Proceedings of the National Academy of Sciences of the United States of America | volume = 109 | issue = 43 | pages = 17442–7 | date = October 2012 | pmid = 23045636 | pmc = 3491489 | doi = 10.1073/pnas.1209000109 | bibcode = 2012PNAS..10917442A | doi-access = free }}</ref>

Several large-scale computational projects, such as [[Rosetta@home]],<ref>{{Cite web|url=http://boinc.bakerlab.org/rosetta/|title=Rosetta@home|website=boinc.bakerlab.org|access-date=14 March 2023}}</ref> [[Folding@home]]<ref>{{Cite web|url=https://foldingathome.org/about-2/the-foldinghome-consortium/|title=The Folding@home Consortium (FAHC) – Folding@home|access-date=14 March 2023}}</ref> and [[Foldit]],<ref>{{Cite web|url=http://fold.it/portal/info/science|title=Foldit|website=fold.it|access-date=14 March 2023}}</ref> target protein folding.

Long continuous-trajectory simulations have been performed on [[Anton (computer)|Anton]], a massively parallel supercomputer designed and built around custom [[ASIC]]s and interconnects by [[D. E. Shaw Research]]. The longest published result of a simulation performed using Anton as of 2011 was a 2.936 millisecond simulation of NTL9 at 355 K.<ref name="pmid22034434">{{cite journal | vauthors = Lindorff-Larsen K, Piana S, Dror RO, Shaw DE | title = How fast-folding proteins fold | journal = Science | volume = 334 | issue = 6055 | pages = 517–20 | date = October 2011 | pmid = 22034434 | doi = 10.1126/science.1208351 | bibcode = 2011Sci...334..517L | s2cid = 27988268 }}</ref> Such simulations are currently able to unfold and refold small proteins (<150 amino acids residues) in equilibrium and predict how mutations affect folding kinetics and stability.<ref name="pmid20974152">{{cite journal | vauthors = Piana S, Piana S, Sarkar K, Lindorff-Larsen K, Guo M, Gruebele M, Shaw DE | title = Computational Design and Experimental Testing of the Fastest-Folding β-Sheet Protein | journal = J. Mol. Biol. | volume = 405 | pages = 43–48 | date = 2010 | issue = 1 | doi = 10.1016/j.jmb.2010.10.023 | pmid = 20974152 }}</ref>

In 2020 a team of researchers that used [[AlphaFold]], an [[artificial intelligence]] (AI) protein structure prediction program developed by [[DeepMind]] placed first in [[CASP]], a long-standing structure prediction contest.<ref name=cnbc20201130>{{Cite news |last=Shead|first=Sam |date=2020-11-30 |title=DeepMind solves 50-year-old 'grand challenge' with protein folding A.I. |url=https://www.cnbc.com/2020/11/30/deepmind-solves-protein-folding-grand-challenge-with-alphafold-ai.html |access-date=2020-11-30|website=CNBC|language=en}}</ref> The team achieved a level of accuracy much higher than any other group.<ref name="Stoddart">{{cite journal |last1=Stoddart |first1=Charlotte |title=Structural biology: How proteins got their close-up |journal=Knowable Magazine |date=1 March 2022 |doi=10.1146/knowable-022822-1|s2cid=247206999 |doi-access=free |url=https://knowablemagazine.org/article/living-world/2022/structural-biology-how-proteins-got-their-closeup |access-date=25 March 2022|url-access=subscription }}</ref> It scored above 90% for around two-thirds of the proteins in CASP's [[Global distance test|global distance test (GDT)]], a test that measures the degree of similarity between the structure predicted by a computational program, and the empirical structure determined experimentally in a lab. A score of 100 is considered a complete match, within the distance cutoff used for calculating GDT.<ref name=science20201130>Robert F. Service, [https://www.science.org/content/article/game-has-changed-ai-triumphs-solving-protein-structures 'The game has changed.' AI triumphs at solving protein structures], ''[[Science (magazine)|Science]]'', 30 November 2020</ref>

AlphaFold's protein structure prediction results at CASP were described as "transformational" and "astounding".<ref name=Callaway2020>{{cite journal |last1=Callaway |first1=Ewen |title='It will change everything': DeepMind's AI makes gigantic leap in solving protein structures |journal=Nature |date=30 November 2020 |volume=588 |issue=7837 |pages=203–204 |doi=10.1038/d41586-020-03348-4 |pmid=33257889 |bibcode=2020Natur.588..203C |s2cid=227243204 }}</ref><ref>{{cite tweet|user=MoAlQuraishi|number=1333383634649313280|title=CASP14 #s just came out and they're astounding}}</ref> Some researchers noted that the accuracy is not high enough for a third of its predictions, and that it does not reveal the physical mechanism of protein folding for the [[protein folding problem]] to be considered solved.<ref>{{cite web |url=https://www.chemistryworld.com/opinion/behind-the-screens-of-alphafold/4012867.article |title=Behind the screens of AlphaFold |first= Phillip |last= Balls|date=9 December 2020|work=Chemistry World }}</ref> Nevertheless, it is considered a significant achievement in [[computational biology]]<ref name=science20201130/> and great progress towards a decades-old grand challenge of biology, predicting the structure of proteins.<ref name=Callaway2020/>

== See also ==
{{Div col|colwidth=20em}}
* [[Anfinsen's dogma]]
* [[Chevron plot]]
* [[Denaturation midpoint]]
* [[Downhill folding]]
* [[Folding (chemistry)]]
* [[Phi value analysis]]
* [[Potential energy of protein]]
* [[Protein dynamics]]
* [[Protein misfolding cyclic amplification]]
* [[Protein structure prediction software]]
* [[Proteopathy]]
* [[Time-resolved mass spectrometry]]
{{Div col end}}

== References ==
{{reflist}}

== External links ==
* [http://www.worldcommunitygrid.org/research/proteome/overview.do Human Proteome Folding Project]

{{Protein tertiary structure}}
{{Protein topics}}
{{Biomolecular structure}}
{{Posttranslational modification}}

{{Authority control}}

[[Category:Biochemical reactions]]
[[Category:Protein folding| ]]
[[Category:Protein structure]]