An arsenal of methods for the experimental characterization of intrinsically disordered proteins - How to choose and combine them?

In this review, we detail the most common experimental approaches to assess and characterize protein intrinsic structural disorder, with the notable exception of NMR and EPR spectroscopy, two ideally suited approaches that will be described in depth in two other reviews within this special issue. We discuss the advantages, the limitations, as well as the caveats of the various methods. We also describe less common and more demanding approaches that enable achieving further insights into the conformational properties of IDPs. Finally, we present recent developments that have enabled assessment of structural disorder in living cells, and discuss the currently available methods to model IDPs as conformational ensembles.

Regulation of measles virus gene expression by P protein coiled-coil properties.

The polymerase of negative-stranded RNA viruses consists of the large protein (L) and the phosphoprotein (P), the latter serving both as a chaperon and a cofactor for L. We mapped within measles virus (MeV) P the regions responsible for binding and stabilizing L and showed that the coiled-coil multimerization domain (MD) of P is required for gene expression. MeV MD is kinked as a result of the presence of a stammer. Both restoration of the heptad regularity and displacement of the stammer strongly decrease or abrogate activity in a minigenome assay. By contrast, P activity is rather tolerant of substitutions within the stammer. Single substitutions at the "a" or "d" hydrophobic anchor positions with residues of variable hydrophobicity revealed that P functionality requires a narrow range of cohesiveness of its MD. Results collectively indicate that, beyond merely ensuring P oligomerization, the MD finely tunes viral gene expression through its cohesiveness.

Author Correction: Exploring intrinsically disordered proteins in Chlamydomonas reinhardtii.

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Exploring intrinsically disordered proteins in Chlamydomonas reinhardtii.

The content of intrinsically disordered protein (IDP) is related to organism complexity, evolution, and regulation. In the Plantae, despite their high complexity, experimental investigation of IDP content is lacking. We identified by mass spectrometry 682 heat-resistant proteins from the green alga, Chlamydomonas reinhardtii. Using a phosphoproteome database, we found that 331 of these proteins are targets of phosphorylation. We analyzed the flexibility propensity of the heat-resistant proteins and their specific features as well as those of predicted IDPs from the same organism. Their mean percentage of disorder was about 20%. Most of the IDPs (~70%) were addressed to other compartments than mitochondrion and chloroplast. Their amino acid composition was biased compared to other classic IDPs. Their molecular functions were diverse; the predominant ones were nucleic acid binding and unfolded protein binding and the less abundant one was catalytic activity. The most represented proteins were ribosomal proteins, proteins associated to flagella, chaperones and histones. We also found CP12, the only experimental IDP from C. reinhardtii that is referenced in disordered protein database. This is the first experimental investigation of IDPs in C. reinhardtii that also combines in silico analysis.

InSiDDe: A Server for Designing Artificial Disordered Proteins.

InSiDDe (In Silico Disorder Design) is a program for the in silico design of intrinsically disordered proteins of desired length and disorder probability. The latter is assessed using IUPred and spans values ranging from 0.55 to 0.95 with 0.05 increments. One to ten artificial sequences per query, each made of 50 to 200 residues, can be generated by InSiDDe. We describe the rationale used to set up InSiDDe and show that an artificial sequence of 100 residues with an IUPred score of 0.6 designed by InSiDDe could be recombinantly expressed in E. coli at high levels without degradation when fused to a natural molecular recognition element (MoRE). In addition, the artificial fusion protein exhibited the expected behavior in terms of binding modulation of the specific partner recognized by the MoRE. To the best of our knowledge, InSiDDe is the first publicly available software for the design of intrinsically disordered protein (IDP) sequences. InSiDDE is publicly available online.

DisProt 7.0: a major update of the database of disordered proteins.

The Database of Protein Disorder (DisProt, URL: www.disprot.org) has been significantly updated and upgraded since its last major renewal in 2007. The current release holds information on more than 800 entries of IDPs/IDRs, i.e. intrinsically disordered proteins or regions that exist and function without a well-defined three-dimensional structure. We have re-curated previous entries to purge DisProt from conflicting cases, and also upgraded the functional classification scheme to reflect continuous advance in the field in the past 10 years or so. We define IDPs as proteins that are disordered along their entire sequence, i.e. entirely lack structural elements, and IDRs as regions that are at least five consecutive residues without well-defined structure. We base our assessment of disorder strictly on experimental evidence, such as X-ray crystallography and nuclear magnetic resonance (primary techniques) and a broad range of other experimental approaches (secondary techniques). Confident and ambiguous annotations are highlighted separately. DisProt 7.0 presents classified knowledge regarding the experimental characterization and functional annotations of IDPs/IDRs, and is intended to provide an invaluable resource for the research community for a better understanding structural disorder and for developing better computational tools for studying disordered proteins.

The intriguing CP12-like tail of adenylate kinase 3 from Chlamydomonas reinhardtii.

UNLABELLED: Adenylate kinases (ADK) are key enzymes that maintain the energetic balance in cellular compartments by catalyzing the reaction: AMP + ATP↔2 ADP. Here, we analyzed the chloroplast ADK 3 from the green alga, Chlamydomonas reinhardtii for the first time. This enzyme bears a C-terminal extension that is highly similar to the C-terminal end of the intrinsically disordered protein CP12 that plays a major role in the redox regulation of key enzymes of the Calvin-Benson cycle like glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and phosphoribulokinase. The only other known example of a CP12-like extension is found in the GapB isoform of GAPDH, where it is responsible for the autonomous redox regulation of the higher plant A2 B2 GAPDH. In this study, we show that the CP12-like tail is not involved in the redox regulation of ADK 3, but contributes greatly to its stability, and is essential for the post-translational modification of the Cys221 residue by glutathione. This report highlights the fact that the C-terminal part of the CP12 protein can act as a moonlighting, intrinsically disordered module conferring additional capabilities to the proteins to which it is added. ENZYMES: Adenylate kinase (ADK, EC 2.7.4.3) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, EC 1.2.1.13).