Editing Protein folding (section)

== 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>