Intrinsically disordered proteins

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Conformational flexibility in SUMO-1 protein (PDB:1a5r). The central part shows relatively ordered structure. Conversely, the N- and C-terminal regions (left and right, respectively) show ‘intrinsic disorder’, although a short helical region persists in the N-terminal tail. Ten alternative NMR models were morphed. Secondary structure elements: α-helices (red), β-strands (blue arrows). [1]

An intrinsically disordered protein (IDP) is a protein that lacks a fixed or ordered three-dimensional structure.[2][3][4] IDPs cover a spectrum of states from fully unstructured to partially structured and include random coils, (pre-)molten globules, and large multi-domain proteins connected by flexible linkers.

The discovery of IDPs has challenged the traditional protein structure paradigm, that protein function depends on a fixed three-dimensional structure. This dogma has been challenged over the last decades by increasing evidence from various branches of structural biology, suggesting that protein dynamics may be highly relevant for such systems. Despite their lack of stable structure, IDPs are a very large and functionally important class of proteins. In some cases, IDPs can adopt a fixed three-dimensional structure after binding to other macromolecules.


An ensemble of NMR structures of the Thylakoid soluble phosphoprotein TSP9, which shows a largely flexible protein chain.[5]

In the 1930s -1950s, the first protein structures were solved by protein crystallography. These early structures suggested that a fixed three-dimensional structure might be generally required to mediate biological functions of proteins. Already in the 1960s, Levinthal's paradox suggested that the systematic conformational search of a long polypeptide is unlikely to yield a single folded protein structure on biologically relevant timescales (i.e. seconds to minutes). Curiously, for many (small) proteins or protein domains, relatively rapid and efficient refolding can be observed in vitro. As stated in Anfinsen's Dogma (1973), the fixed 3D structure of these proteins is uniquely encoded in its primary structure (=amino acid sequence), is kinetically accessible and stable under a range of (near)physiological conditions and can therefore be considered as the native state of such "ordered" proteins.

During the subsequent decades, however, many large protein regions could not be assigned in x-ray datasets, indicating that they occupy multiple positions which average out in electron density maps. The lack of a fixed, unique positions relative to the crystal lattice suggested that these regions were "disordered". Additional techniques for determining protein structures, such as NMR, demonstrated the presence of large flexible linkers and termini in many solved structural ensembles. It is now generally accepted that proteins exist as an ensemble of similar structures with some regions more constrained than others. Intrinsically Unstructured Proteins (IUPs) occupy the extreme end of this spectrum of flexibility, whereas IDPs also include proteins of considerable local structure tendency or flexible multidomain assemblies.

These disordered regions have subsequently been shown to have important functions both in vitro and in vivo. In the 2000s, bioinformatic predictions of intrinsic disorder in proteins indicated that intrinsic disorder is more common in sequenced/predicted proteomes than in known structures in the protein database.[2] Based on DISOPRED2 prediction, long (>30 residue) disordered segments occur in 2.0% of archaean, 4.2% of eubacterial and 33.0% of eukaryotic proteins.[6] In the 2010s it became clear that IDPs are highly abundant among disease-related proteins.[7]

Biological roles of intrinsic disorder[edit]

Many disordered proteins have the binding affinity with their receptors regulated by post-translational modification, thus it has been proposed that the flexibility of disordered proteins facilitates the different conformational requirements for binding the modifying enzymes as well as their receptors.[8] Intrinsic disorder is particularly enriched in proteins implicated in cell signaling, transcription and chromatin remodeling functions.[9][10]

Flexible linkers[edit]

Disordered regions are often found as flexible linkers (or loops) connecting two globular or transmembrane domains. Linker sequences vary greatly in length and amino acid sequence, but are similar in amino acid composition (rich in polar uncharged amino acids). Flexible linkers allow the connecting domains to freely twist and rotate through space to recruit their binding partners or for those binding partners to induce larger scale interdomain conformation changes.

Linear motifs[edit]

Linear motifs are short disordered segments of proteins that mediate functional interactions with other proteins or other biomolecules (RNA, DNA, sugars etc.). Many roles or linear motifs are associated with cell regulation, for instance in control of cell shape, subcellular localisation of individual proteins and regulated protein turnover. Often, post-translational modifications such as phosphorylation tune the affinity (not rarely by several orders of magnitude) of individual linear motifs for specific interactions. Relatively rapid evolution and a relatively small number of structural restraints for establishing novel (low-affinity) interfaces make it particularly challenging to detect linar motifs but their widespread biological roles and the fact that many viruses mimick/hijack linear motifs to efficiently recode infected cells underlines the timely urgency of research on this very challenging and exciting topic.

Coupled folding and binding[edit]

Many unstructured proteins undergo transitions to more ordered states upon binding to their targets. The coupled folding and binding may be local, involving only a few interacting residues, or it might involve an entire protein domain. It was recently shown that the coupled folding and binding allows the burial of a large surface area that would be possible only for fully structured proteins if they were much larger.[11] Moreover, certain disordered regions might serve as "molecular switches" in regulating certain biological function by switching to ordered conformation upon molecular recognition like small molecule-binding, DNA/RNA binding, ion interactions etc.[12]

The ability of disordered proteins to bind, and thus to exert a function, shows that stability is not a required condition. Many short functional sites, for example Short Linear Motifs are over-represented in disordered proteins.

Disorder in the bound state (fuzzy complexes)[edit]

Intrinsically disordered proteins can retain their conformational freedom even when they bind specifically to other proteins. The structural disorder in bound state can be static or dynamic. In fuzzy complexes structural multiplicity is required for function and the manipulation of the bound disordered region changes activity. The conformational ensemble of the complex is modulated via post-translational modifications or protein interactions.[13] Specificity of DNA binding proteins often depends on the length of fuzzy regions, which is varied by alternative splicing.[14]

Disorder identification and analysis[edit]

Disorder prediction software[edit]

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 VMD. Blue and red arrows point to missing residues on receptor and growth hormone, respectively.

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.

Intrinsically unstructured proteins are characterized by a low content of bulky hydrophobic amino acids and a high proportion of polar and charged amino acids. Thus disordered sequences cannot bury sufficient hydrophobic core to fold like stable globular proteins. In some cases, hydrophobic clusters in disordered sequences provide the clues for identifying the regions that undergo coupled folding and binding. Such signatures are the basis of the prediction methods below.

Many disordered proteins also reveal low complexity sequences, i.e. sequences with over-representation of a few residues. 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. There are many computational methods that exploit sequence information to predict whether a protein is disordered.[15] Notable examples of such software include IUPRED and Disopred. Different software may use different definitions of disorder. Since the methods above use different definitions of disorder and they were trained on different datasets, it is difficult to estimate their relative accuracy. 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 PDB files as REMARK465, missing electron densities in X-ray structures).

Experimental validation[edit]

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 NMR spectroscopy. The lack of electron density in 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 nm) circular dichroism (esp. a pronounced minimum at ~200 nm) or infrared spectroscopy. Unfolded proteins also have exposed backbone peptide groups exposed to solvent, so that they are readily cleaved by proteases, undergo rapid hydrogen-deuterium exchange and exhibit a small dispersion (<1 ppm) in their 1H amide chemical shifts as measured by 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.[16][17] 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. [18]

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[19] to study conformational flexibilty of IDPs and the kinetics of structural transitions, optical tweezers[20] for high-resolution insights into the ensembles of IDPs and their oligomers or aggregates, nanopores[21] to reveal global shape distributions of IDPs, magnetic tweezers[22] to study structural transitions for long times at low forces, high-speed AFM[23] to visualise the spatio-temporal flexibility of IDPs directly.

Disorder and disease[edit]

Intrinsically unstructured proteins have been implicated in a number of diseases.[24] Aggregation of misfolded proteins is the cause of many synucleinopathies. The aggregation of the intrinsically unstructured protein 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.[25] Many key oncogenes have large intrinsically unstructured regions, for example p53 and BRCA1. These regions of the proteins are responsible for mediating many of their interactions.

Computer simulations[edit]

Structural and dynamical properties of intrinsically unstructured proteins are being studied by molecular dynamics simulations.[26][27][28] Findings from these simulations suggest a highly flexible conformational ensemble of intrinsically disordered proteins at different temperatures which is related to the presence of low free energy barriers.

Effects of confinement have been also recently addressed.[29] These studies suggest that confinement tends to increase the population of turn structures with respect to the population of coils and β-hairpins for instance.

Morover, various protocols and methodologies of analysis of IDP's such as studies based on quantitative analysis of GC content in genes and their respective chromosomal bands to understand functionally Intrinsically disordered protein segments.[30][31]

Pioneering IDP research labs[edit]

More and more research laboratories start to study protein disorder, both using experimental techinques (e.g. SAXS-NMR, single-molecule fluorescence) and computationally (prediction of protein disorder).

See also[edit]


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