Proteine intrinsecamente disordinate
{{T|inglese|biologia|marzo 2018|argomento 2=chimica}}{{C|la traduzione sembra non cogliere il senso (neppure a livello linguistico) della voce inglese da cui è tratta|biologia|maggio 2024|argomento2=chimica}}{{Correggere|biologia|maggio 2024|la prosa risente di una traduzione difettosa dall'inglese|argomento2=chimica}}[[File:1a5r SUMO-1 protein.gif|thumb|upright=1.4|Flessibilità conformazionale nella proteina [[SUMO protein|SUMO-1]] (PDB:[http://www.rcsb.org/pdb/explore/explore.do?structureId=1a5r 1a5r]). 
La parte centrale mostra la struttura relativamente ordinata. Invece, le regioni N- e C-terminali (rispettivamente a sinistra e a destra) mostrano un 'disordine intrinseco', anche se una breve regione elicoidale persiste nella coda N-terminale. <!--Ten alternative [[Nuclear magnetic resonance spectroscopy of proteins|NMR models]] were morphed.--> Elementi della struttura secondaria: l'[[alfa elica]] in rosso e il [[foglietto  β]] in azzurro.
<ref>{{Cita libro |coautori=Majorek K, Kozlowski L, Jakalski M, Bujnicki, JM |curatore-cognome=Bujnicki |curatore-nome=J |titolo=Prediction of Protein Structures, Functions, and Interactions |editore=John Wiley & Sons, Ltd. |data=18 dicembre 2008 |pp=39–62 |doi=10.1002/9780470741894.ch2 |url=ftp://genesilico.pl/lukaskoz/pdfs/Majorek.pdf |10= |capitolo=Chapter 2: First Steps of Protein Structure Prediction |isbn=978-0-470-51767-3 |urlmorto=sì }}</ref>]]

Una '''proteina intrinsecamente disordinata''' ('''IDP''') è una [[proteine|proteina]] a cui manca una [[struttura terziaria]] fissa o ordinata, tipicamente in assenza di partner macromolecolari con cui interagire, come altre proteine o [[RNA]]. <ref name="pmid11381529">{{Cita pubblicazione|coautori= Dunker AK, Lawson JD, Brown CJ, Williams RM, Romero P, Oh JS, Oldfield CJ, Campen AM, Ratliff CM, Hipps KW, Ausio J, Nissen MS, Reeves R, Kang C, Kissinger CR, Bailey RW, Griswold MD, Chiu W, Garner EC, Obradovic Z |titolo= Intrinsically disordered protein |rivista= Journal of Molecular Graphics & Modelling |volume= 19 |numero= 1 |pp= 26–59 |anno= 2001 | pmid = 11381529 | doi = 10.1016/s1093-3263(00)00138-8 }}</ref><ref>{{Cita pubblicazione|coautori= Dyson HJ, Wright PE |titolo= Intrinsically unstructured proteins and their functions |rivista= Nature Reviews. Molecular Cell Biology |volume= 6 |numero= 3 |pp= 197–208 |data= March 2005 | pmid = 15738986 | doi = 10.1038/nrm1589 }}</ref><ref name="struct08">{{Cita pubblicazione|coautori= Dunker AK, Silman I, Uversky VN, Sussman JL |titolo= Function and structure of inherently disordered proteins |rivista= Current Opinion in Structural Biology |volume= 18 |numero= 6 |pp= 756–64 |data= December 2008 | pmid = 18952168 | doi = 10.1016/j.sbi.2008.10.002 }}</ref> Gli stati con cui si presentano le IDP coprono un ampio spettro, da completamente non strutturati a parzialmente strutturati, compresi quelli con spire casuali, aggregati simili a [[globulo fuso|globuli fusi]], e linker flessibili in grandi proteine [[Dominio (biochimica)|multi-dominio]]. A volte sono considerate come una classe separata di proteine, insieme alle [[proteina globulare|quelle globulari]], [[Proteina fibrosa|fibrose]] e di [[proteina di membrana|membrana]]).<ref>{{Cita pubblicazione|coautori= Andreeva A, Howorth D, Chothia C, Kulesha E, Murzin AG |titolo= SCOP2 prototype: a new approach to protein structure mining |rivista= Nucleic Acids Research |volume= 42 |numero= Database issue |pp= D310-4 |data= January 2014 | pmid = 24293656 | pmc = 3964979 | doi = 10.1093/nar/gkt1242 }}</ref>

La scoperta delle IDP ha cambiato il [[paradigma]] della struttura tradizionale delle proteine,  la cui funzione dipende da una [[Struttura terziaria|struttura tridimensionale ordinata]]. Questo dogma è stato messo in discussione negli anni 2000 e 2010, con l'aumento di prove da vari rami della biologia strutturale, che suggeriscono che la dinamica delle proteine potrebbe essere molto rilevante per tali sistemi. Nonostante la mancanza di una struttura stabile, le IDP sono una classe di proteine vastissima e funzionalmente importante. In alcuni casi, le IDP possono adottare una struttura tridimensionale fissa a seguito di legame con altre macromolecole. Nel complesso, le IDP sono in molti modi diverse dalle proteine strutturate e tendono ad avere proprietà distintive in termini di funzione, struttura, sequenza, interazioni, evoluzione e regolazione. <ref>{{Cita pubblicazione|coautori= van der Lee R, Buljan M, Lang B, Weatheritt RJ, Daughdrill GW, Dunker AK, Fuxreiter M, Gough J, Gsponer J, Jones DT, Kim PM, Kriwacki RW, Oldfield CJ, Pappu RV, Tompa P, Uversky VN, Wright PE, Babu MM |titolo= Classification of intrinsically disordered regions and proteins |rivista= Chemical Reviews |volume= 114 |numero= 13 |pp= 6589–631 |data= July 2014 | pmid = 24773235 | pmc = 4095912 | doi = 10.1021/cr400525m }}</ref>

== Storia ==
[[File:PDB 2fft EBI.jpg|thumb|Un insieme di strutture [[Risonanza magnetica nucleare|NMR]] della fosfoproteina solubile [[tilacoide]] TSP9, che mostra una catena proteica ampiamente flessibile. <ref name="pmid17176085">{{Cita pubblicazione|coautori= Song J, Lee MS, Carlberg I, Vener AV, Markley JL |titolo= Micelle-induced folding of spinach thylakoid soluble phosphoprotein of 9 kDa and its functional implications |rivista= Biochemistry |volume= 45 |numero= 51 |pp= 15633–43 |data= December 2006 | pmid = 17176085 | pmc = 2533273 | doi = 10.1021/bi062148m }}</ref>|250px]]
Dagli anni '30 agli anni '50, le prime [[struttura proteica|strutture proteiche]] furono risolte e scoperte grazie alla [[cristallografia]]. Queste prime strutture suggerivano che, per mediare le funzioni biologiche delle proteine, dovesse essere necessaria, in genere, una [[struttura terziaria|struttura tridimensionale]] fissa. Quando affermarono che le proteine hanno una sola configurazione definita in modo univoco, Mirsky e Pauling non riconobbero che il lavoro di Fisher avrebbe sostenuto la loro tesi con il modello "chiave-serratura" (1894). Queste pubblicazioni consolidarono il [[dogma centrale della biologia molecolare]] in quanto la sequenza determina la struttura che, a sua volta, determina la funzione delle proteine. Nel 1950, Karush scrisse di "adattabilità configurazionale", contraddicendo tutte le ipotesi e le ricerche del diciannovesimo secolo. Era convinto che le proteine avessero più di una configurazione allo stesso livello di energia e potessero sceglierne una nel momento in cui si legavano ad altri substrati. Negli anni '60, il [[paradosso di Levinthal]] suggerì che la ricerca conformazionale sistematica di un polipeptide lungo non è in grado di produrre una singola struttura proteica piegata su scale temporali biologicamente rilevanti (cioè da secondi a minuti). Curiosamente, per molte (piccole) proteine, o domini proteici, è possibile osservare in vitro un ripiegamento relativamente rapido ed efficiente. Come affermato nel [[dogma di Anfinsen]] del 1973, la struttura tridimensionale di queste proteine è codificata in modo univoco nella sua struttura primaria (la sequenza amminoacidica), è cineticamente accessibile e stabile in una gamma di condizioni fisiologiche (simili) e può quindi essere considerata come lo stato nativo di tali proteine "ordinate".

Durante i decenni successivi, tuttavia, non è stato possibile assegnare numerose regioni di proteine nelle serie di dati [[Cristallografia a raggi X|ottenuti ai raggi X]], il che indicava che esse occupano più posizioni intermedie nelle mappe di [[densità elettronica]]. La mancanza di posizioni fisse e uniche rispetto al [[reticolo cristallino]] suggeriva che queste regioni erano "disordinate". La [[Spettroscopia di risonanza magnetica nucleare|spettroscopia a risonanza magnetica nucleare]] delle proteine ha anche dimostrato la presenza di grandi linker (o congiuntori) e terminazioni flessibili in molti complessi strutturali. 

È ormai generalmente accettato che le proteine esistano come un insieme di strutture simili, con alcune regioni più vincolate  di altre. Le proteine intrinsecamente non strutturate (IUP) occupano l'estremo di questo spettro di flessibilità e comprendono anche proteine con una notevole tendenza alla struttura locale o ad assemblaggi flessibili multidominio. Queste regioni altamente disordinate di proteine sono state in seguito collegate a fenomeni funzionalmente importanti come la [[regolazione allosterica]] e la [[catalisi enzimatica]].
<ref name="pmid21570668">{{Cita pubblicazione|coautori= Bu Z, Callaway DJ |titolo= Proteins move! Protein dynamics and long-range allostery in cell signaling |rivista= Advances in Protein Chemistry and Structural Biology |volume= 83 |pp= 163–221 |anno= 2011 | pmid = 21570668 | doi = 10.1016/B978-0-12-381262-9.00005-7 | isbn = 978-0-12-381262-9 |serie= Advances in Protein Chemistry and Structural Biology }}</ref>
<ref name="pmid20099310">{{Cita pubblicazione|coautori= Kamerlin SC, Warshel A |titolo= At the dawn of the 21st century: Is dynamics the missing link for understanding enzyme catalysis? |rivista= Proteins |volume= 78 |numero= 6 |pp= 1339–75 |data= May 2010 | pmid = 20099310 | pmc = 2841229 | doi = 10.1002/prot.22654 }}</ref>

Il disordine intrinseco è particolarmente elevato tra le proteine che regolano la cromatina e la trascrizione e le previsioni [[bioinformatica|bioinformatiche]] indicano che esso è più comune nei [[Genoma|genomi]] e nei [[proteoma|proteomi]] sequenziati rispetto a strutture note nel database delle proteine. Sulla base della previsione DISOPRED2, segmenti disordinati lunghi (> 30 residui) si verificano nel 2,0% di archae, nel 4,2% di eubatteri e nel 33,0% delle proteine eucariote.<ref>{{Cita pubblicazione|coautori= Ward JJ, Sodhi JS, McGuffin LJ, Buxton BF, Jones DT |titolo= Prediction and functional analysis of native disorder in proteins from the three kingdoms of life |rivista= Journal of Molecular Biology |volume= 337 |numero= 3 |pp= 635–45 |data= March 2004 | pmid = 15019783 | doi = 10.1016/j.jmb.2004.02.002 }}</ref> Nel 2001, in un articolo intitolato ''Intrinsically disordered protein'', ha messo in dubbio che le informazioni trovate di recente fossero state ignorate per 50 anni.<ref>{{Cita pubblicazione|coautori= Dunker AK, Lawson JD, Brown CJ, Williams RM, Romero P, Oh JS, Oldfield CJ, Campen AM, Ratliff CM, Hipps KW, Ausio J, Nissen MS, Reeves R, Kang C, Kissinger CR, Bailey RW, Griswold MD, Chiu W, Garner EC, Obradovic Z |titolo= Intrinsically disordered protein |rivista= Journal of Molecular Graphics & Modelling |volume= 19 |numero= 1 |pp= 26–59 |data= 1º gennaio 2001 | pmid = 11381529 | doi = 10.1016/s1093-3263(00)00138-8 }}</ref>

Nel 2010 è diventato chiaro che le IDP sono molto abbondanti tra le proteine correlate a malattie, come [[alfa-sinucleina]] e [[Proteina Tau|tau]].<ref>{{Cita pubblicazione|coautori= Uversky VN, Oldfield CJ, Dunker AK |titolo= Intrinsically disordered proteins in human diseases: introducing the D2 concept |rivista= Annual Review of Biophysics |volume= 37 |pp= 215–46 |anno= 2008 | pmid = 18573080 | doi = 10.1146/annurev.biophys.37.032807.125924 }}</ref>

==Ruoli biologici==
Molte proteine disordinate hanno l'affinità di legame con i loro recettori regolata dalla [[modificazione post-traduzionale]]: per questo motivo, è stato proposto che la flessibilità delle proteine disordinate faciliti i diversi requisiti conformazionali per legare gli enzimi modificanti e i loro recettori.<ref>{{Cita pubblicazione|coautori= Collins MO, Yu L, Campuzano I, Grant SG, Choudhary JS |titolo= Phosphoproteomic analysis of the mouse brain cytosol reveals a predominance of protein phosphorylation in regions of intrinsic sequence disorder |rivista= Molecular & Cellular Proteomics |volume= 7 |numero= 7 |pp= 1331–48 |data= July 2008 | pmid = 18388127 | doi = 10.1074/mcp.M700564-MCP200 }}</ref> Il disordine intrinseco è particolarmente arricchito nelle proteine implicate nella[[Trascrizione (biologia)|trascrizione]] e nella [[segnalazione cellulare]], nonché nella trascrizione e nel rimodellamento della [[cromatina]].<ref>{{Cita pubblicazione|coautori= Iakoucheva LM, Brown CJ, Lawson JD, Obradović Z, Dunker AK |titolo= Intrinsic disorder in cell-signaling and cancer-associated proteins |rivista= Journal of Molecular Biology |volume= 323 |numero= 3 |pp= 573–84 |data= October 2002 | pmid = 12381310 | doi = 10.1016/S0022-2836(02)00969-5 }}</ref><ref>{{Cita pubblicazione|coautori= Sandhu KS |titolo= Intrinsic disorder explains diverse nuclear roles of chromatin remodeling proteins |rivista= Journal of Molecular Recognition |volume= 22 |numero= 1 |pp= 1–8 |anno= 2009 | pmid = 18802931 | doi = 10.1002/jmr.915 }}</ref>

===Legami flessibili===
Le regioni disordinate si trovano spesso come linker flessibili o ad anello che collegano dominii. Le sequenze dei linker variano notevolmente in lunghezza ma sono tipicamente ricche di [[amminoacido|amminoacidi]] polari neutri (senza carica). I linker flessibili consentono ai domini di connessione di ruotare e torcersi liberamente per reclutare i loro compagni di legame attraverso la [[Dinamica proteica|dinamica]] del dominio proteico. Consentono inoltre ai loro compagni di legame di indurre cambiamenti conformazionali su larga scala mediante [[regolazione allosterica]] a lungo raggio<ref name="pmid21570668" /><ref name="pmid11381529" />

===Motivi lineari===
I motivi lineari sono brevi segmenti disordinati di proteine che mediano le interazioni funzionali con altre proteine o altre biomolecole (RNA, DNA, zuccheri ecc.). Molti compiti dei motivi lineari sono associati alla regolazione cellulare, ad esempio nel controllo della forma cellulare, nella localizzazione subcellulare delle singole proteine e nel turnover proteico regolato. Spesso, le modificazioni post-traduzionali come la fosforilazione sintonizzano l'affinità (non raramente per diversi ordini di grandezza) dei singoli motivi lineari per specifiche interazioni. L’evoluzione relativamente rapida e un numero relativamente piccolo di restrizioni strutturali per stabilire nuove interfacce (a bassa affinità) rendono particolarmente difficile individuare motivi lineari ma i loro ruoli biologici diffusi e il fatto che molti virus imitino / sequestrino motivi lineari per ricodificare efficientemente le cellule infette sottolinea la tempestiva urgenza della ricerca su questo argomento molto stimolante ed emozionante. A differenza delle proteine globulari, le IDP non hanno tasche attive disposte nello spazio. Tuttavia, nell'80% delle IDP (~ 3 dozzine) sottoposte a caratterizzazione strutturale dettagliata mediante NMR ci sono motivi lineari denominati PreSMos (motivi pre-strutturati) che sono elementi strutturali secondari transitori innescati per il riconoscimento del bersaglio. In diversi casi è stato dimostrato che queste strutture transitorie diventano strutture secondarie piene e stabili, ad esempio eliche, sul legame del bersaglio. Quindi, I PreSMos sono i siti putativi attivi nelle IDP<ref>{{Cita pubblicazione|coautori= Lee SH, Kim DH, Han JJ, Cha EJ, Lim JE, Cho YJ, Lee C, Han KH |titolo= Understanding pre-structured motifs (PreSMos) in intrinsically unfolded proteins |rivista= Current Protein & Peptide Science |volume= 13 |numero= 1 |pp= 34–54 |data= February 2012 | pmid = 22044148 | doi = 10.2174/138920312799277974 }}</ref>.
             
===Ripiegamento e legame===
Molte proteine non strutturate subiscono transizioni verso stati più ordinati al momento del legame ai loro bersagli (ad esempio le [[MoRFs]]<ref name=ref2006>{{Cita pubblicazione|coautori= Mohan A, Oldfield CJ, Radivojac P, Vacic V, Cortese MS, Dunker AK, Uversky VN |titolo= Analysis of molecular recognition features (MoRFs) |rivista= Journal of Molecular Biology |volume= 362 |numero= 5 |pp= 1043–59 |data= October 2006 | pmid = 16935303 | doi = 10.1016/j.jmb.2006.07.087 }}</ref>). Il ripiegamento e il legame accoppiati possono essere locali, coinvolgendo solo pochi residui, oppure potrebbero coinvolgere un intero dominio proteico. Recentemente è stato dimostrato che la ripiegatura e il legame accoppiati consentono la copertura di un'ampia superficie che sarebbe possibile solo per proteine completamente strutturate se fossero molto più grandi.<ref>{{Cita pubblicazione|coautori= Gunasekaran K, Tsai CJ, Kumar S, Zanuy D, Nussinov R |titolo= Extended disordered proteins: targeting function with less scaffold |rivista= Trends in Biochemical Sciences |volume= 28 |numero= 2 |pp= 81–5 |data= February 2003 | pmid = 12575995 | doi = 10.1016/S0968-0004(03)00003-3 }}</ref> Inoltre, alcune regioni disordinate potrebbero servire come "interruttori molecolari" nella regolazione di determinate funzioni biologiche passando alla conformazione ordinata al momento del riconoscimento molecolare come legami di piccole molecole, legami DNA / RNA, interazioni ioniche ecc.<ref>{{Cita pubblicazione|coautori= Sandhu KS, Dash D |titolo= Dynamic alpha-helices: conformations that do not conform |rivista= Proteins |volume= 68 |numero= 1 |pp= 109–22 |data= July 2007 | pmid = 17407165 | doi = 10.1002/prot.21328 }}</ref>

La capacità delle proteine disordinate di legarsi, e quindi di esercitare una funzione, dimostra che la stabilità non è una condizione richiesta. Molti siti funzionali brevi sono sovrarappresentati nelle proteine disordinate.

=== Disordine nello stato limite (fuzzy complexes) ===
Le proteine intrinsecamente disordinate possono mantenere la loro libertà conformazionale anche quando si legano specificamente ad altre proteine. Il disordine strutturale nello stato legato può essere statico o dinamico.               <!--In [[fuzzy complex]]es structural multiplicity is required for function and the manipulation of the bound disordered region changes activity. The [[conformational ensembles|conformational ensemble]] of the complex is modulated via post-translational modifications or protein interactions.<ref>{{cite journal | vauthors = Fuxreiter M | title = Fuzziness: linking regulation to protein dynamics | journal = Molecular bioSystems | volume = 8 | issue = 1 | pages = 168–77 | date = January 2012 | pmid = 21927770 | doi = 10.1039/c1mb05234a }}</ref> Specificity of DNA binding proteins often depends on the length of fuzzy regions, which is varied by alternative splicing.<ref>{{cite journal | vauthors = Fuxreiter M, Simon I, Bondos S | title = Dynamic protein-DNA recognition: beyond what can be seen | journal = Trends in Biochemical Sciences | volume = 36 | issue = 8 | pages = 415–23 | date = August 2011 | pmid = 21620710 | doi = 10.1016/j.tibs.2011.04.006 }}</ref>

==Structural aspects==
Intrinsically disordered proteins adapt many different structures in vivo according to the cell's conditions, creating a structural or conformational ensemble.<ref name="uversky11">{{cite journal | vauthors = Uversky VN | title = Intrinsically disordered proteins from A to Z | journal = The International Journal of Biochemistry & Cell Biology | volume = 43 | issue = 8 | pages = 1090–103 | date = August 2011 | pmid = 21501695 | doi = 10.1016/j.biocel.2011.04.001 }}</ref><ref name="oldfield14">{{Cite journal |year=2014 |author1=Oldfield |first1= C. |title=Intrinsically Disordered Proteins and Intrinsically Disordered Protein Regions| journal = Annual Review of Biochemistry | volume = 83 | pages = 553–584 |doi=10.1146/annurev-biochem-072711}}</ref>

Therefore, their structures are strongly function-related. However, only few proteins are fully disordered in their native state. Disorder is mostly found in intrinsically disordered regions (IDRs) within an otherwise well-structured protein. The term intrinsically disordered protein (IDP) therefore includes proteins that contain IDRs as well as fully disordered proteins.

The existence and kind of protein disorder is encoded in its amino acid sequence.<ref name="pmid11381529" /> In general, IDPs are characterized by a low content of bulky [[hydrophobic]] amino acids and a high proportion of polar and charged amino acids, usually referred to as low hydrophobicity.<ref name="uversky11" /> This property leads to good interactions with water. Furthermore, high net charges promote disorder because of electrostatic repulsion resulting from equally charged residues.<ref name="oldfield14" /> Thus disordered sequences cannot sufficiently bury a hydrophobic core to fold into stable globular proteins. In some cases, hydrophobic clusters in disordered sequences provide the clues for identifying the regions that undergo coupled folding and binding (refer to [[intrinsically disordered proteins#biological roles|biological roles]]).
Many disordered proteins reveal regions without any regular secondary structure These regions can be termed as flexible, compared to structured loops. While the latter are rigid and contain only one set of Ramachandran angles, IDPs involve multiple sets of angles.<ref name="oldfield14" /> The term flexibility is also used for well-structured proteins, but describes a different phenomenon in the context of disordered proteins. Flexibility in structured proteins is bound to an equilibrium state, while it is not so in IDPs.<ref name="oldfield14" />
Many disordered proteins also reveal [[low complexity sequences]], i.e. sequences with over-representation of a few [[residue (chemistry)|residue]]s. While low complexity sequences are a strong indication of disorder, the reverse is not necessarily true, that is, not all disordered proteins have low complexity sequences. Disordered proteins have a low content of predicted [[secondary structure]].

==Experimental validation==
Intrinsically unfolded proteins, once purified, can be identified by various experimental methods. The primary method to obtain information on disordered regions of a protein is [[Protein NMR|NMR spectroscopy]]. The lack of electron density in [[X-ray crystallography|X-ray crystallographic]] studies may also be a sign of disorder.

Folded proteins have a high density (partial specific volume of 0.72-0.74 mL/g) and commensurately small [[radius of gyration]]. Hence, unfolded proteins can be detected by methods that are sensitive to molecular size, density or [[hydrodynamic drag]], such as [[size exclusion chromatography]], [[analytical ultracentrifugation]], [[small angle X-ray scattering (SAXS)]], and measurements of the [[diffusion constant]]. Unfolded proteins are also characterized by their lack of [[secondary structure]], as assessed by far-UV (170-250&nbsp;nm) [[circular dichroism]] (esp. a pronounced minimum at ~200&nbsp;nm) or [[infrared]] spectroscopy. Unfolded proteins also have exposed backbone [[peptide]] groups exposed to solvent, so that they are readily cleaved by [[protease]]s, undergo rapid [[hydrogen-deuterium exchange]] and exhibit a small dispersion (<1 ppm) in their 1H amide [[chemical shift]]s as measured by [[protein NMR|NMR]]. (Folded proteins typically show dispersions as large as 5 ppm for the amide protons.) 
Recently, new methods including [[Fast parallel proteolysis (FASTpp)]] have been introduced, which allow to determine the fraction folded/disordered without the need for purification.<ref name="fastpp">{{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 | pages = e46147 | year = 2012 | pmid = 23056252 | pmc = 3463568 | doi = 10.1371/journal.pone.0046147 | editor1-last = Uversky | editor1-first = Vladimir N }}</ref><ref name="methods">{{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 }}</ref> Even subtle differences in the stability of missense mutations, protein partner binding and (self)polymerisation-induced folding of (e.g.) coiled-coils can be detected using FASTpp as recently demonstrated using the tropomyosin-troponin protein interaction.<ref name="Robaszkiewicz2015">{{cite journal | vauthors = Robaszkiewicz K, Ostrowska Z, Cyranka-Czaja A, Moraczewska J | title = Impaired tropomyosin-troponin interactions reduce activation of the actin thin filament | journal = Biochimica et Biophysica Acta | volume = 1854 | issue = 5 | pages = 381–90 | date = May 2015 | pmid = 25603119 | doi = 10.1016/j.bbapap.2015.01.004 }}</ref> Fully unstructured protein regions can be experimentally validated by their hypersusceptibility to proteolysis using short digestion times and low protease concentrations.<ref name="APC-Axin-proteolysis">{{cite journal | vauthors = Minde DP, Radli M, Forneris F, Maurice MM, Rüdiger SG | title = Large extent of disorder in Adenomatous Polyposis Coli offers a strategy to guard Wnt signalling against point mutations | journal = PloS One | volume = 8 | issue = 10 | pages = e77257 | year = 2013 | pmid = 24130866 | pmc = 3793970 | doi = 10.1371/journal.pone.0077257 | editor1-last = Buckle | editor1-first = Ashley M }}</ref>

Bulk methods to study IDP structure and dynamics include [[SAXS]] for ensemble shape information, [[NMR]] for atomistic ensemble refinement, [[Fluorescence]] for visualising molecular interactions and conformational transitions, x-ray crystallography to highlight more mobile regions in otherwise rigid protein crystals, cryo-EM to reveal less fixed parts of proteins, light scattering to monitor size distributions of IDPs or their aggregation kinetics, [[Circular Dichroism]] to monitor secondary structure of IDPs.

Single-molecule methods to study IDPs include spFRET<ref>{{cite journal | vauthors = Brucale M, Schuler B, Samorì B | title = Single-molecule studies of intrinsically disordered proteins | journal = Chemical Reviews | volume = 114 | issue = 6 | pages = 3281–317 | date = March 2014 | pmid = 24432838 | doi = 10.1021/cr400297g }}</ref> to study conformational flexibility of IDPs and the kinetics of structural transitions, [[optical tweezers]]<ref name=Neupane2014>{{cite journal | vauthors = Neupane K, Solanki A, Sosova I, Belov M, Woodside MT | title = Diverse metastable structures formed by small oligomers of α-synuclein probed by force spectroscopy | journal = PloS One | volume = 9 | issue = 1 | pages = e86495 | year = 2014 | pmid = 24475132 | pmc = 3901707 | doi = 10.1371/journal.pone.0086495 }}</ref> for high-resolution insights into the ensembles of IDPs and their oligomers or aggregates, nanopores<ref>{{cite journal | vauthors = Japrung D, Dogan J, Freedman KJ, Nadzeyka A, Bauerdick S, Albrecht T, Kim MJ, Jemth P, Edel JB | title = Single-molecule studies of intrinsically disordered proteins using solid-state nanopores | journal = Analytical Chemistry | volume = 85 | issue = 4 | pages = 2449–56 | date = February 2013 | pmid = 23327569 | doi = 10.1021/ac3035025 }}</ref> to reveal global shape distributions of IDPs, magnetic tweezers<ref>{{cite journal | vauthors = Min D, Kim K, Hyeon C, Cho YH, Shin YK, Yoon TY | title = Mechanical unzipping and rezipping of a single SNARE complex reveals hysteresis as a force-generating mechanism | journal = Nature Communications | volume = 4 | issue = 4 | pages = 1705 | year = 2013 | pmid = 23591872 | pmc = 3644077 | doi = 10.1038/ncomms2692 }}</ref> to study structural transitions for long times at low forces, high-speed [[Atomic force microscope|AFM]]<ref>{{cite journal | vauthors = Miyagi A, Tsunaka Y, Uchihashi T, Mayanagi K, Hirose S, Morikawa K, Ando T | title = Visualization of intrinsically disordered regions of proteins by high-speed atomic force microscopy | journal = Chemphyschem | volume = 9 | issue = 13 | pages = 1859–66 | date = September 2008 | pmid = 18698566 | doi = 10.1002/cphc.200800210 }}</ref> to visualise the spatio-temporal flexibility of IDPs directly.

==Disorder prediction==

[[File:Remark465 1a22 HUMAN GROWTH HORMONE BOUND TO SINGLE RECEPTOR.png|thumb|REMARK465 - missing electron densities in X-ray structure representing protein disorder ({{PDB|1a22}}, human growth hormone bound to receptor). Compilation of screenshots from PDB database and molecule representation via [[Visual Molecular Dynamics|VMD]]. Blue and red arrows point to missing residues on receptor and growth hormone, respectively.|250px]]

[[List of disorder prediction software|Disorder prediction algorithms]] can predict Intrinsic Disorder (ID) propensity with high accuracy (approaching around 80%) based on primary sequence composition, similarity to unassigned segments in protein x-ray datasets, flexible regions in NMR studies and physico-chemical properties of amino acids.

===Distinguishing IDPs from well-structured proteins===

Separating disordered from ordered proteins is essential for disorder prediction.
One of the first steps to find a factor that distinguishes IDPs from non-IDPs is to specify biases within the amino acid composition. The following hydrophilic, charged amino acids A, R, G, Q, S, P, E and K have been characterized as disorder-promoting amino acids, while order-promoting amino acids W, C, F, I, Y, V, L, and N are hydrophobic and uncharged. The remaining amino acids H, M, T and D are ambiguous, found in both ordered and unstructured regions.<ref name="pmid11381529" /> This information is the basis of most sequence-based predictors.
Regions with little to no secondary structure, also known as NORS (NO Regular Secondary structure) regions,<ref>{{cite journal | vauthors = Schlessinger A, Schaefer C, Vicedo E, Schmidberger M, Punta M, Rost B | title = Protein disorder--a breakthrough invention of evolution? | journal = Current Opinion in Structural Biology | volume = 21 | issue = 3 | pages = 412–8 | date = June 2011 | pmid = 21514145 | doi = 10.1016/j.sbi.2011.03.14 }}</ref> and low-complexity regions can easily be detected. However, not all disordered proteins contain such low complexity sequences.

===Prediction methods===
{{Main article|List of disorder prediction software}}

Determining disordered regions from biochemical methods is very costly and time-consuming. Due to the variable nature of IDPs, only certain aspects of their structure can be detected, so that a full characterization requires a large number of different methods and experiments. This further increases the expense of IDP determination. In order to overcome this obstacle, computer-based methods are created for predicting protein structure and function. It is one of the main goals of bioinformatics to derive knowledge by prediction. Predictors for IDP function are also being developed, but mainly use structural information such as [[#linear motifs|linear motif]] sites.<ref name="struct08" /><ref>{{Cite journal |year=2011 |author1=Tompa |first1= P. |title=Unstructural biology coming of age | journal = Current Opinion in Structural Biology | volume = 21 | pages = 419–425 |doi=10.1016/j.sbi.2011.03.12}}</ref> There are different approaches for predicting IDP structure, such as [[artificial neural network|neural networks]] or matrix calculations, based on different structural and/or biophysical properties.

Many computational methods exploit sequence information to predict whether a protein is disordered.<ref>{{cite journal | vauthors = Ferron F, Longhi S, Canard B, Karlin D | title = A practical overview of protein disorder prediction methods | journal = Proteins | volume = 65 | issue = 1 | pages = 1–14 | date = October 2006 | pmid = 16856179 | doi = 10.1002/prot.21075 }}</ref> Notable examples of such software include IUPRED and Disopred. Different methods may use different definitions of disorder. Meta-predictors show a new concept, combining different primary predictors to create a more competent and exact predictor.

Due to the different approaches of predicting disordered proteins, estimating their relative accuracy is fairly difficult. For example, [[artificial neural network|neural networks]] are often trained on different datasets. The disorder prediction category is a part of biannual [[CASP]] experiment that is designed to test methods according accuracy in finding regions with missing 3D structure (marked in [[Protein Data Bank (file format)|PDB files]] as REMARK465, missing electron densities in X-ray structures).

== Disorder and disease ==
Intrinsically unstructured proteins have been implicated in a number of diseases.<ref name="d2d2 concept">{{cite journal | vauthors = Uversky VN, Oldfield CJ, Dunker AK | title = Intrinsically disordered proteins in human diseases: introducing the D2 concept | journal = Annual Review of Biophysics | volume = 37 | pages = 215–46 | year = 2008 | pmid = 18573080 | doi = 10.1146/annurev.biophys.37.032807.125924 }}</ref> Aggregation of misfolded proteins is the cause of many [[synuclein]]opathies and toxicity as those proteins start binding to each other randomly and can lead to cancer or cardiovascular diseases. Thereby, misfolding can happen spontaneously because millions of copies of proteins are made during the lifetime of an organism. The aggregation of the intrinsically unstructured protein [[Alpha-synuclein|α-synuclein]] is thought to be responsible. The structural flexibility of this protein together with its susceptibility to modification in the cell leads to misfolding and aggregation. Genetics, oxidative and nitrative stress as well as mitochondrial impairment impact the structural flexibility of the unstructured α-synuclein protein and associated disease mechanisms.<ref name="Genetic mutation">{{cite journal | vauthors = Wise-Scira O, Dunn A, Aloglu AK, Sakallioglu IT, Coskuner O | title = Structures of the E46K mutant-type α-synuclein protein and impact of E46K mutation on the structures of the wild-type α-synuclein protein | journal = ACS Chemical Neuroscience | volume = 4 | issue = 3 | pages = 498–508 | date = March 2013 | pmid = 23374074 | doi = 10.1021/cn3002027 }}</ref> Many key [[tumour suppressor]]s have large intrinsically unstructured regions, for example p53 and BRCA1. These regions of the proteins are responsible for mediating many of their interactions. Taking the cell's native defense mechanisms as a model drugs can be developed, trying to block the place of noxious substrates and inhibiting them, and thus counteracting the disease.<ref>{{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 }}</ref>

==Computer simulations==
Owing to high structural heterogeneity, NMR/SAXS experimental parameters obtained will be an average over a large number of highly diverse and disordered states (an ensemble of disordered states). Hence, to understand the structural implications of these experimental parameters, there is a necessity for accurate representation of these ensembles by computer simulations. All-atom molecular dynamic simulations can be used for this purpose but their use is limited by the accuracy of current force-fields in representing disordered proteins. Nevertheless, some force-fields have been explicitly developed for studying disordered proteins by optimising force-field parameters using available NMR data for disordered proteins. (examples are CHARMM 22*, CHARMM 32<ref>{{cite journal | vauthors = Best RB, Zhu X, Shim J, Lopes PE, Mittal J, Feig M, Mackerell AD | title = Optimization of the additive CHARMM all-atom protein force field targeting improved sampling of the backbone φ, ψ and side-chain χ(1) and χ(2) dihedral angles | journal = Journal of Chemical Theory and Computation | volume = 8 | issue = 9 | pages = 3257–3273 | date = September 2012 | pmid = 23341755 | pmc = 3549273 | doi = 10.1021/ct300400x }}</ref>, Amber ff03* etc.)

MD simulations restrained by experimental parameters (restrained-MD) have also been used to characterise disordered proteins.<ref>{{cite journal | vauthors = Best RB | title = Computational and theoretical advances in studies of intrinsically disordered proteins | journal = Current Opinion in Structural Biology | volume = 42 | pages = 147–154 | date = February 2017 | pmid = 28259050 | doi = 10.1016/j.sbi.2017.01.006 }}</ref><ref>{{cite journal | vauthors = Chong SH, Chatterjee P, Ham S | title = Computer Simulations of Intrinsically Disordered Proteins | journal = Annual Review of Physical Chemistry | volume = 68 | pages = 117–134 | date = May 2017 | pmid = 28226222 | doi = 10.1146/annurev-physchem-052516-050843 }}</ref><ref>{{cite journal | vauthors = Fox SJ, Kannan S | title = Probing the dynamics of disorder | journal = Progress in Biophysics and Molecular Biology | volume = 128 | pages = 57–62 | date = September 2017 | pmid = 28554553 | doi = 10.1016/j.pbiomolbio.2017.05.008 }}</ref> In principle, one can sample the whole conformational space given an MD simulation (with accurate Force-field) is run long enough. Because of very high structural heterogeneity, the time scales that needs to be run for this purpose are very large and are limited by computational power. However, other computational techniques such as accelerated-MD simulations,<ref>{{cite journal | vauthors = Terakawa T, Takada S | title = Multiscale ensemble modeling of intrinsically disordered proteins: p53 N-terminal domain | journal = Biophysical Journal | volume = 101 | issue = 6 | pages = 1450–8 | date = September 2011 | pmid = 21943426 | pmc = 3177054 | doi = 10.1016/j.bpj.2011.08.003 | bibcode = 2011BpJ...101.1450T }}</ref> or [[replica exchange]] simulations<ref name="fisher2011">{{cite journal|doi=10.1016/j.sbi.2011.04.001|pmid=21530234|title=Constructing ensembles for intrinsically disordered proteins|journal=Current Opinion in Structural Biology|volume=21|issue=3|pages=426|year=2011|last1=Fisher|first1=Charles K|last2=Stultz|first2=Collin M}}</ref><ref>{{cite journal | vauthors = Apicella A, Marascio M, Colangelo V, Soncini M, Gautieri A, Plummer CJ | title = Molecular dynamics simulations of the intrinsically disordered protein amelogenin | journal = Journal of Biomolecular Structure & Dynamics | volume = 35 | issue = 8 | pages = 1813–1823 | date = June 2017 | pmid = 27366858 | doi = 10.1080/07391102.2016.1196151 }}</ref> or [[metadynamics]]<ref name="zerze2015">{{cite journal | vauthors = Zerze GH, Miller CM, Granata D, Mittal J | title = Free energy surface of an intrinsically disordered protein: comparison between temperature replica exchange molecular dynamics and bias-exchange metadynamics | journal = Journal of Chemical Theory and Computation | volume = 11 | issue = 6 | pages = 2776–82 | date = June 2015 | pmid = 26575570 | doi = 10.1021/acs.jctc.5b00047 }}</ref><ref name="granata2015">{{cite journal | vauthors = Granata D, Baftizadeh F, Habchi J, Galvagnion C, De Simone A, Camilloni C, Laio A, Vendruscolo M | title = The inverted free energy landscape of an intrinsically disordered peptide by simulations and experiments | journal = Scientific Reports | volume = 5 | pages = 15449 | date = October 2015 | pmid = 26498066 | pmc = 4620491 | doi = 10.1038/srep15449 | bibcode = 2015NatSR...515449G }}</ref> have been used to sample broader conformational space in smaller time scales. 

Moreover, various protocols and methods of analyzing IDPs, such as studies based on quantitative analysis of GC content in genes and their respective chromosomal bands, have been used to understand functional IDP segments.<ref>{{cite journal | vauthors = Uversky VN | title = Digested disorder: Quarterly intrinsic disorder digest (January/February/March, 2013) | journal = Intrinsically Disordered Proteins | volume = 1 | issue = 1 | pages = e25496 | year = 2013 | pmid = 28516015 | pmc = 5424799 | doi = 10.4161/idp.25496 }}</ref><ref>{{cite journal | vauthors = Costantini S, Sharma A, Raucci R, Costantini M, Autiero I, Colonna G | title = Genealogy of an ancient protein family: the Sirtuins, a family of disordered members | journal = BMC Evolutionary Biology | volume = 13 | pages = 60 | date = March 2013 | pmid = 23497088 | pmc = 3599600 | doi = 10.1186/1471-2148-13-60 }}</ref>

== See also ==

* [[IDPbyNMR]]
* [[DisProt|DisProt database]]
* [[MobiDB|MobiDB database]]
* [[Molten globule]]
* [[Random coil]]-->

== Note ==
<references/>

== Collegamenti esterni ==
* [http://proteopedia.org/wiki/index.php/Intrinsically_Disordered_Protein Intrinsically disordered protein at Proteopedia]
* [http://mobidb.bio.unipd.it MobiDB: a comprehensive database or intrinsic protein disorder annotations]
* [http://www.ideal.force.cs.is.nagoya-u.ac.jp/IDEAL/ IDEAL - '''I'''ntrinsically '''D'''isordered proteins with '''E'''xtensive '''A'''nnotations and '''L'''iterature] {{Webarchive|url=https://web.archive.org/web/20200502160132/http://www.ideal.force.cs.is.nagoya-u.ac.jp/IDEAL/ |date=2 maggio 2020 }}
* [http://d2p2.pro/ D<sup>2</sup>P<sup>2</sup> Database of Disordered Protein Predictions]
* [https://web.archive.org/web/20180304192615/http://iimcb.genesilico.pl/metadisorder/protein_disorder_intrinsically_unstructured_proteins_gallery_images.html Gallery of images of intrinsically disordered proteins]
* [http://www.tandfonline.com/toc/kidp20/current#.VcZObfntlBf First IDP journal covering all topics of IDP research]
* [http://www.landesbioscience.com/journals/IDP IDP Journal]
* [http://www.disprot.org/ Database of experimentally validated IDPs]
* [https://web.archive.org/web/20180310010556/http://pedb.vib.be/ IDP ensemble database]
{{Portale|Biologia|Chimica}}
[[Categoria:Proteine]]