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Conformational analysis: An approach to the prediction of RDCs from disordered protein chains, integrating the effect of nearest neighbors and the alignment characteristics of the statistical coil, is reported. NMR residual dipolar couplings (RDC) are sensitive probes of conformational sampling in unfolded proteins (see picture). [ABSTRACT FROM AUTHOR]

Konformationsanalyse: Ein Ansatz zur Vorhersage von residualen dipolaren NMR ‐ Kopplungskonstanten (RDCs) für ungeordnete Proteinketten, der sowohl den Effekt der nächsten Nachbarn als auch die Ausrichtung des statistischen Knäuels berücksichtigt, wird vorgestellt. RDC ‐ Werte sind empfindliche Sonden zum Abtasten der Konformationen ungefalteter Proteine (siehe Bild). [ABSTRACT FROM AUTHOR]

The detailed, quantitative characterization of unfolded proteins is a largely unresolved task due to the enormous experimental and theoretical difficulties in describing the highly dimensional space of their conformational ensembles. Recently, residual dipolar coupling (RDC) and paramagnetic relaxation enhancement (PRE) data have provided large numbers of experimental parameters on unfolded states. To obtain a minimal model of the unfolded state according to such data we have developed new modules for the use of steric alignment RDCs and PREs as constraints in ensemble structure calculations by the program XPLOR-NIH. As an example, ensemble calculations were carried out on urea-denatured ubiquitin using a total of 419 previously obtained RDCs and 253 newly determined PREs from eight cysteine mutants coupled to MTSL. The results show that only a small number of about 10 conformers is necessary to fully reproduce the experimental RDCs, PREs and average radius of gyration. Cα contacts determined on a large set (400) of 10-conformer ensembles show significant (10-20%) populations of conformations that are similar to ubiquitin's A-state, i.e. corresponding to an intact native first β-hairpin and α-helix as well as non-native a-helical conformations in the C-terminal half. Thus, methanol/acid (A-state) and urea denaturation lead to similar low energy states of the protein ensemble, presumably due to the weakening of the hydrophobic core. Similar contacts are obtained in calculations using solely RDCs or PREs. The sampling statistics of the Cα contacts in the ensembles follow a simple binomial distribution. It follows that the present RDC, PRE, and computational methods allow the statistically significant detection of subconformations in the unfolded ensemble at population levels of a few percent. [ABSTRACT FROM AUTHOR]

Abstract: We present a study of the denaturation of a truncated, cycle3 variant of green fluorescent protein (GFP). Chemical denaturation is used to unfold the protein, with changes in structure being monitored by the green fluorescence, tyrosine fluorescence and far-UV circular dichroism. The results show that the denaturation behaviour of GFP is complex compared to many small proteins: equilibrium is established only very slowly, over the time course of weeks, suggesting that there are high folding/unfolding energy barriers. Unfolding kinetics confirm that the rates of unfolding at low concentrations of denaturant are very low, consistent with the slow establishment of the equilibrium. In addition, we find that GFP significantly populates an intermediate state under equilibrium conditions, which is compact and stable with respect to the unfolded state (m IU =4.6 kcal mol−1 M−1 and ΔG IU =12.5 kcal mol−1). The global and local stability of GFP was probed further by measuring the hydrogen/deuterium (H/D) NMR exchange rates of more than 157 assigned amide protons. Analysis at two different values of pH showed that amide protons within the β-barrel structure exchange at the EX2 limit, consequently, free energies of exchange could be calculated and compared to those obtained from the denaturation-curve studies providing further support for the three-state model and the existence of a stable intermediate state. Analysis reveals that amide protons in β-strands 7, 8, 9 and 10 have, on average, higher exchange rates than others in the β-barrel, suggesting that there is greater flexibility in this region of the protein. Forty or so amide protons were found which do not undergo significant exchange even after several months and these are clustered into a core region encompassing most of the β-strands, at least at one end of the barrel structure. It is likely that these residues play an important role in stabilizing the structure of the intermediate state. The intermediate state observed in the chemical denaturation studies described here, is similar to that observed at pH 4 in other studies. [Copyright &y& Elsevier]

RNA-binding proteins (RBPs) have intrinsically disordered regions (IDRs) whose biophysical properties have yet to be explored to the same extent as those of the folded RNA interacting domains. These IDRs are essential to the formation of biomolecular condensates, such as stress and RNA granules, but dysregulated assembly can be pathological. Because of their structural heterogeneity, IDRs are best studied by NMR spectroscopy. In this study, we used NMR spectroscopy to investigate the structural propensity and self-association of the IDR of the RBP Musashi-1. We identified two transient α-helical regions (residues ~208–218 and ~270–284 in the IDR, the latter with a polyalanine tract). Strong NMR line broadening in these regions and circular dichroism and micrography data suggest that the two α-helical elements and the hydrophobic residues in between may contribute to the formation of oligomers found in stress granules and implicated in Alzheimer's disease. Bioinformatics analysis suggests that polyalanine stretches in the IDRs of RBPs may have evolved to promote RBP assembly. [ABSTRACT FROM AUTHOR]

NMR spectroscopy is a powerful method for studying disordered proteins, providing atomic resolution and ensemble‐averaged information. In their Communication on page 687 ff., M. Blackledge et al. show that by analyzing local and long‐range effects, residual dipolar couplings can be determined up to six orders of magnitude faster than by existing techniques. [ABSTRACT FROM AUTHOR]

NMR ‐ Spektroskopie ist eine leistungsstarke Methode zur Untersuchung ungeordneter Proteine, die atomare Auflösung und über das Ensemble gemittelte Information liefert. In ihrer Zuschrift auf S. 715 ff. zeigen M. Blackledge et al., dass durch Analyse von lokalen und weitreichenden Effekten dipolare Restkopplungen um bis zu sechs Größenordnungen schneller bestimmt werden können als mit existierenden Methoden. [ABSTRACT FROM AUTHOR]

Paralogs, arising from gene duplications, increase the functional diversity of proteins. Protein functions in paralog families have been extensively studied, but little is known about the roles that intrinsically disordered regions (IDRs) play in their paralogs. Without a folded structure to restrain them, IDRs mutate more diversely along with evolution. However, how the diversity of IDRs in a paralog family affects their functions is unexplored. Using the RNA-binding protein Musashi family as an example, we applied multiple structural techniques and phylogenetic analysis to show how members in a paralog family have evolved their IDRs to different physicochemical properties but converge to the same function. In this example, the lower prion-like tendency of Musashi-1's IDRs, rather than Musashi-2's, is compensated by its higher α-helical propensity to assist their assembly. Our work suggests that, no matter how diverse they become, IDRs could evolve different traits to a converged function, such as liquid-liquid phase separation. A comparison of structure-function relationship for intrinsically disordered regions (IDRs) among paralogs shows that differences in physicochemical properties of the IDR between paralogs are compensated for to converge on the same biophysical function. [ABSTRACT FROM AUTHOR]

The article reports on the recent developments in the interpretation of experimental nuclear magnetic resonance (NMR) spectroscopy data to describe the free-energy landscape explored by intrinsically disordered proteins (IDPs) and intrinsically disordered protein regions (IDPRs). It states that NMR spectroscopy is among numerous techniques suitable for structural characterization of IDPs and IDPRs in solution. The report also tackles analytical approaches to describe free-energy landscape of IDPs and IDPRs.

Current NMR information on side-chain conformations of unfolded protein states is sparse due to the poor dispersion particularly of side-chain proton resonances. We present here optimized schemes for the detection of 3JHαHβ, 3JNHβ and 3JC'Hβ scalar and 1DC'lHβ residual dipolar couplings (RDCs) in unfolded proteins. For urea-denatured ubiquitin and protein G, up to six 3J-couplings to 1Hβ are detected, which define the ϰ1 angle at very high precision. Interpretation of the 3Jcouplings by a model of mixed staggered ϰ1 rotamers yields excellent agreement and also provides stereoassignments for 1Hβ methylene protons. For all observed amino acids with the exception of leucine, the chemical shift of 1H133 protons was found downfield from 1Hβ. For most residues, the precision of individual ϰ1 rotamer populations is better than 2%. The experimental ϰ1 rotamer populations are in the vicinity of averages obtained from coil regions in folded protein structures. However, individual variations from these averages of up to 40% are highly significant and indicate sequence- and residue-specific interactions. Particularly strong deviations from the coil average are found for serine and threonine residues, an effect that may be explained by a weakening of side-chain to backbone hydrogen bonds in the urea-denatured state. The measured 1DCβHβ RDCs correlate well with predicted RDCs that were calculated from a sterically aligned coil model ensemble and the 3J-derived Xi rotamer populations. This agreement supports the coil model as a good first approximation of the unfolded state. Deviations between measured and predicted values at certain sequence locations indicate that the description of the local backbone conformations can be improved by incorporation of the RDC information. The ease of detection of a large number of highly precise side-chain RDCs opens the possibility for a more rigorous characterization of both side-chain and backbone conformations in unfolded proteins. [ABSTRACT FROM AUTHOR]

Purinosomes serve as metabolons to enhance de novo purine synthesis (DNPS) efficiency through compartmentalizing DNPS enzymes during stressed conditions. However, the mechanism underpinning purinosome assembly and its pathophysiological functions remains elusive. Here, we show that K6-polyubiquitination of the DNPS enzyme phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazolesuccinocarboxamide synthetase (PAICS) by cullin-5/ankyrin repeat and SOCS box containing 11 (Cul5/ASB11)-based ubiquitin ligase plays a driving role in purinosome assembly. Upon several purinosome-inducing cues, ASB11 is upregulated by relieving the H3K9me3/HP1α-mediated transcriptional silencing, thus stimulating PAICS polyubiquitination. The polyubiquitinated PAICS recruits ubiquitin-associated protein 2 (UBAP2), a ubiquitin-binding protein with multiple stretches of intrinsically disordered regions, thereby inducing phase separation to trigger purinosome assembly for enhancing DNPS pathway flux. In human melanoma, ASB11 is highly expressed to facilitate a constitutive purinosome formation to which melanoma cells are addicted for supporting their proliferation, viability, and tumorigenesis in a xenograft model. Our study identifies a driving mechanism for purinosome assembly in response to cellular stresses and uncovers the impact of purinosome formation on human malignancies. [Display omitted] • Stresses that increase purine demand upregulate ASB11 by epigenetic mechanisms • ASB11 promotes PAICS ubiquitination to recruit the ubiquitin-binding protein UBAP2 • UBAP2 governs multivalent interactions to drive purinosome assembly • Melanoma cells form purinosome constitutively to support their proliferation and survival In this study, Chou et al. discovered that PAICS polyubiquitination induced by cell stresses triggers the recruitment of UBAP2 to confer multivalent interactions for driving phase separation to form purinosome condensates. Melanoma cells are addicted to the constitutive purinosome formation to supply sufficient purines for their proliferation and survival. [ABSTRACT FROM AUTHOR]

The TAR DNA-binding protein of 43 kDa (TDP-43) has been identified as the main component of amyotrophic lateral sclerosis (ALS) cytoplasmic inclusions. The link between this proteinopathy and TDP-43′s intrinsically disordered C-terminal domain is well known, but recently also, this domain has been shown to be involved in the formation of the membraneless organelles that mediate TDP-43′s functions. The mechanisms that underpin the liquid-liquid phase separation (LLPS) of these membraneless organelles undergo remain elusive. Crucially though, these factors may be the key to understanding the delicate balance between TDP-43′s physiological and pathological functions. In this study, we used nuclear magnetic resonance spectroscopy and optical methods to demonstrate that an α-helical component in the centre (residues 320–340) of the C-terminal domain is related to the protein's self-association and LLPS. Systematically analysing ALS-related TDP-43 mutants (G298S, M337V, and Q331K) in different buffer conditions at different temperatures, we prove that this phase separation is driven by hydrophobic interactions but is inhibited by electrostatic repulsion. Based on these findings, we rationally introduced a mutant, W334G, and demonstrate that this mutant disrupts LLPS without disturbing this α-helical propensity. This tryptophan may serve as a key residue in this protein's LLPS. [ABSTRACT FROM AUTHOR]

Motivation: Intrinsically disordered proteins (IDPs) represent a significant fraction of the human proteome. The classical structure function paradigm that has successfully underpinned our understanding of molecular biology breaks down when considering proteins that have no stable tertiary structure in their functional form. One convenient approach is to describe the protein in terms of an equilibrium of rapidly inter-converting conformers. Currently, tools to generate such ensemble descriptions are extremely rare, and poorly adapted to the prediction of experimental data.Results: We present flexible-meccano—a highly efficient algorithm that generates ensembles of molecules, on the basis of amino acid-specific conformational potentials and volume exclusion. Conformational sampling depends uniquely on the primary sequence, with the possibility of introducing additional local or long-range conformational propensities at an amino acid-specific resolution. The algorithm can also be used to calculate expected values of experimental parameters measured at atomic or molecular resolution, such as nuclear magnetic resonance (NMR) and small angle scattering, respectively. We envisage that flexible-meccano will be useful for researchers who wish to compare experimental data with those expected from a fully disordered protein, researchers who see experimental evidence of deviation from ‘random coil’ behaviour in their protein, or researchers who are interested in working with a broad ensemble of conformers representing the flexibility of the IDP of interest.Availability: A fully documented multi-platform executable is provided, with examples, at http://www.ibs.fr/science-213/scientific-output/software/flexible-meccano/Contact: martin.blackledge@ibs.fr [ABSTRACT FROM PUBLISHER]

During oxidative folding, the formation of disulfide bonds has profound effects on guiding the protein folding pathway. Until now, comparatively little is known about the changes in the conformational dynamics in folding intermediates of proteins that contain only a subset of their native disulfide bonds. In this comprehensive study, we probe the conformational landscape of non-native states of lysozyme containing a single native disulfide bond utilizing nuclear magnetic resonance (NMR) spectroscopy, small-angle X-ray scattering (SAXS), circular dichroism (CD) data, and modeling approaches. The impact on conformational dynamics varies widely depending on the loop size of the single disulfide variants and deviates significantly from random coil predictions for both NMR and SAXS data. From these experiments, we conclude that the introduction of single disulfides spanning a large portion of the polypeptide chain shifts the structure and dynamics of hydrophobic core residues of the protein so that these regions exhibit levels of order comparable to the native state on the nanosecond time scale. [ABSTRACT FROM AUTHOR]

Most biological functions involve protein–protein interactions. Our understanding of these interactions is based mainly on those of structured proteins, because encounters between intrinsically disordered proteins (IDPs) or proteins with intrinsically disordered regions (IDRs) are much less studied, regardless of the fact that more than half eukaryotic proteins contain IDRs. RNA-binding proteins (RBPs) are a large family whose members almost all have IDRs in addition to RNA binding domains. These IDRs, having low sequence similarity, interact, but structural details on these interactions are still lacking. Here, using the IDRs of two RBPs (hnRNA-A2 and TDP-43) as a model, we demonstrate that the rate at which TDP-43′s IDR undergoes the neurodegenerative disease related α-helix-to-β-sheet transition increases in relation to the amount of hnRNP-A2′s IDR that is present. There are more than 1500 RBPs in human cells and most of them have IDRs. RBPs often join the same complexes to regulate genes. In addition to the structured RNA-recognition motifs, our study demonstrates a general mechanism through which RBPs may regulate each other's functions through their IDRs. [ABSTRACT FROM AUTHOR]