Intrinsically unstructured proteins

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An ensemble of NMR structures of the Thylakoid soluble phosphoprotein TSP9, which shows a largely flexible protein chain.[1]

Intrinsically unstructured proteins, often referred to as naturally unfolded proteins or disordered proteins, are proteins characterized by lack of stable tertiary structure when the protein exists as an isolated polypeptide chain (a subunit) under physiological conditions in vitro.[2][3] The discovery of intrinsically unfolded proteins challenged the traditional protein structure paradigm, which states that a specific well-defined structure was required for the correct function of a protein and that the structure defines the function of the protein. This is clearly not the case for intrinsically unfolded proteins that remain functional despite the lack of a well-defined structure. Such proteins, in some cases, can adopt a fixed three dimensional structure after binding to other macromolecules.

Biological role of intrinsic disorder

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.[4] Intrinsic disorder is particularly enriched in proteins implicated in cell signaling, transcription and chromatin remodeling functions.[5][6]

Flexible linkers

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.

Coupled folding and binding

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.[7] 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.[8]

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 bound state (Fuzzy complexes)

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.[9] Specificity of DNA binding proteins often depends on the length of fuzzy regions, which is varied by alternative splicing.[10]

Sequence signatures of disorder

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 overrepresentation 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.

Experimental identification of intrinsically unstructured proteins

Intrinsically unfolded proteins, once purified, can be identified by various experimental methods. 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 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.)

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.

Recently, new methods including Fast parallel proteolysis (FASTpp) have been introduced, which allow to determine the fraction folded/disordered without the need for purification.[11][12]

Disorder prediction software

Main article: List of disorder prediction software

There are many computational methods that exploit sequence information to predict whether a protein is disordered.[13] 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). 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.[14][15]

Disorder and disease

Intrinsically unstructured proteins have been implicated in a number of diseases.[16] 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.

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.

The significance of protein disorder in disease was stated as D2 (disorder in disorders) concept.

Computer Simulations

Structural and dynamical properties of intrinsically unstructured proteins are being studied by molecular dynamics simulations.[17][18][19] 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.[20] 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.

See also

References

  1. ↑ Song J, Lee MS, Carlberg I, Vener AV, Markley JL (December 2006). "Micelle-induced folding of spinach thylakoid soluble phosphoprotein of 9 kDa and its functional implications". Biochemistry 45 (51): 15633–43. doi:10.1021/bi062148m. PMC 2533273. PMID 17176085. 
  2. ↑ Dyson HJ, Wright PE (March 2005). "Intrinsically unstructured proteins and their functions". Nat. Rev. Mol. Cell Biol. 6 (3): 197–208. doi:10.1038/nrm1589. PMID 15738986. 
  3. ↑ Dunker AK, Silman I, Uversky VN, Sussman JL (December 2008). "Function and structure of inherently disordered proteins". Curr. Opin. Struct. Biol. 18 (6): 756–64. doi:10.1016/j.sbi.2008.10.002. PMID 18952168. 
  4. ↑ Collins MO, Yu L, Campuzano I, Grant SG, Choudhary JS (July 2008). "Phosphoproteomic analysis of the mouse brain cytosol reveals a predominance of protein phosphorylation in regions of intrinsic sequence disorder". Mol. Cell Proteomics 7 (7): 1331–48. doi:10.1074/mcp.M700564-MCP200. PMID 18388127. 
  5. ↑ Iakoucheva LM, Brown CJ, Lawson JD, Obradović Z, Dunker AK (October 2002). "Intrinsic disorder in cell-signaling and cancer-associated proteins". J. Mol. Biol. 323 (3): 573–84. doi:10.1016/S0022-2836(02)00969-5. PMID 12381310. 
  6. ↑ Sandhu KS (2009). "Intrinsic disorder explains diverse nuclear roles of chromatin remodeling proteins". J. Mol. Recognit. 22 (1): 1–8. doi:10.1002/jmr.915. PMID 18802931. 
  7. ↑ Gunasekaran K, Tsai CJ, Kumar S, Zanuy D, Nussinov R (February 2003). "Extended disordered proteins: targeting function with less scaffold". Trends Biochem. Sci. 28 (2): 81–5. doi:10.1016/S0968-0004(03)00003-3. PMID 12575995. 
  8. ↑ Sandhu KS, Dash D (July 2007). "Dynamic alpha-helices: conformations that do not conform". Proteins 68 (1): 109–22. doi:10.1002/prot.21328. PMID 17407165. 
  9. ↑ Fuxreiter, M (2012). "Fuzziness: Linking regulation to protein dynamics". Molecular bioSystems 8 (1): 168–77. doi:10.1039/c1mb05234a. PMID 21927770. 
  10. ↑ Fuxreiter, M; Simon, I; Bondos, S (2011). "Dynamic protein-DNA recognition: Beyond what can be seen". Trends in biochemical sciences 36 (8): 415–23. doi:10.1016/j.tibs.2011.04.006. PMID 21620710. 
  11. ↑ Minde, David P.; Maurice, Madelon M.; Rüdiger, Stefan G. D. (2012). "Determining Biophysical Protein Stability in Lysates by a Fast Proteolysis Assay, FASTpp". In Uversky, Vladimir N. PLoS ONE 7 (10): e46147. doi:10.1371/journal.pone.0046147. PMC 3463568. PMID 23056252. 
  12. ↑ Park, C; Marqusee, S (2005). "Pulse proteolysis: A simple method for quantitative determination of protein stability and ligand binding". Nature methods 2 (3): 207–12. doi:10.1038/nmeth740. PMID 15782190. 
  13. ↑ Ferron F, Longhi S, Canard B, Karlin D (October 2006). "A practical overview of protein disorder prediction methods". Proteins 65 (1): 1–14. doi:10.1002/prot.21075. PMID 16856179. 
  14. ↑ Uversky, Vladimir N (2013). "Digested disorder: Quarterly intrinsic disorder digest (January/February/March, 2013)". Intrinsically Disordered Proteins 1: e25496. doi:10.4161/idp.25496. 
  15. ↑ Susan Costantini, Ankush Sharma, Raffaele Raucci, Maria Costantini, Ida Autiero, and Giovanni Colonna (March 2013). "Genealogy of an ancient protein family: the Sirtuins, a family of disordered members". BMC evolutionary biology 13: 60. doi:10.1186/1471-2148-13-60. PMC 3599600. PMID 23497088. 
  16. ↑ Uversky, Vladimir N.; Oldfield, Christopher J.; Dunker, A. Keith (2008). "Intrinsically Disordered Proteins in Human Diseases: Introducing the D2Concept". Annual Review of Biophysics 37: 215–46. doi:10.1146/annurev.biophys.37.032807.125924. PMID 18573080. 
  17. ↑ Mittal J, Yoo T, Georgiou G, Truskett T (December 2013). "Structural Ensemble of an Intrinsically Disordered Polypeptide". J. Of Phys. Chem B 117: 118–124. doi:10.1021/jp308984e. 
  18. ↑ Ojeda-May P, Pu J (August 2013). "Replica exchange molecular dynamics simulations of an α/β-type small acid soluble protein (SASP)". Biophysical Chemistry 184: 17–21. doi:10.1016/j.bpc.2013.07.014. PMID 24029407. 
  19. ↑ Higo J, Ito H, Kuroda M, Ono S, Nakajima N, Nakamura H (March 2001). "Energy landscape of a peptide consisting of α-helix, 3-10-helix, β-hairpin, and other disordered conformations". Protein Science 10 (6): 1160–1171. doi:10.1110/ps.44901. PMC 2374007. PMID 11369854. 
  20. ↑ Rao J, Cruz L (March 2013). "Effects of Confinement on the Structure and Dynamics of an Intrinsically Disordered Peptide: A Molecular-Dynamics Study". J. Of Phys. Chem B 117 (14): 3707–3719. doi:10.1021/jp310623x. 
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