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Conserved residues in protein homolog sequence alignments are structurally or functionally important. For intrinsically disordered proteins (IDPs) or proteins with intrinsically disordered regions (IDRs), however, alignment often fails because they lack a steric structure to constrain evolution. Although sequences vary, the physicochemical features of IDRs may be preserved in maintaining function. Therefore, a method to retrieve common IDR features may help identify functionally important residues. We applied un-supervised contrastive learning to train a model with self-attention neuronal networks on human IDR orthologs. During training, parameters were optimized to match sequences in ortholog pairs but not in other IDRs. The trained model successfully identifies previously reported critical residues from experimental studies, especially those with an overall pattern (e.g. multiple aromatic residues or charged blocks) rather than short motifs. This predictive model can therefore be used to identify potentially important residues in other proteins.Availability and implementationThe training scripts are available on GitHub (https://github.com/allmwh/IFF). The training datasets have been deposited in an Open Science Framework repository (https://osf.io/jk29b). The trained model can be run from the Jupyter Notebook in the GitHub repository using Binder (mybinder.org). The only required input is the primary sequence.

Aromatic residues appeared relatively late in the evolution of protein sequences to stabilize the globular proteins' folding core and are less in the intrinsically disordered regions (IDRs). Recent advances in protein liquid–liquid phase separation (LLPS) studies have also shown that aromatic residues in IDRs often act as “stickers” to promote multivalent interactions in forming higher‐order oligomers. To study how general these structure‐promoting residues are in IDRs, we compared levels of sequence disorder in RNA binding proteins (RBPs), which are often found to undergo LLPS, and the human proteome. We found that aromatic residues appear more frequently than expected in the IDRs of RBPs and, through multiple sequence alignment analysis, those aromatic residues are often conserved among chordates. Using TDP‐43, FUS, and some other well‐studied LLPS proteins as examples, the conserved aromatic residues are important to their LLPS‐related functions. These analyses suggest that aromatic residues may have contributed twice to evolution: stabilizing structured proteins and assembling biomolecular condensates.

Aromatic residues appeared relatively late in the evolution of protein sequences. They stabilize the hydrophobic core of globular proteins and are typically absent from intrinsically disordered regions (IDRs). However, recent advances in protein liquid-liquid phase separation (LLPS) studies have shown that aromatic residues in IDRs often act as important “stickers”, promoting multivalent interactions and the formation of higher-order oligomers. To reconcile this apparent contradiction, we compared levels of sequence disorder in RNA binding proteins and the human proteome and found that aromatic residues appear more frequently than expected in the IDRs of RNA binding proteins, which are often found to undergo LLPS. Phylogenetic analysis shows that aromatic residues are highly conserved among chordates, highlighting their importance in LLPS-driven functional assembly. These results suggest therefore that aromatic residues have contributed twice to evolution: in stabilizing structured proteins and in the assembly of biomolecular condensates.

Background and aims: Post-tuberculosis bronchial stenosis (PTBS) is one of the most common complications of tracheobronchial tuberculosis. Silicone stent serves as a major treatment for maintaining airway patency. However, silicone stent placement remains a large challenge in patients with severe cicatricial PTBS. Our objective was to evaluate the efficacy and safety of covered, self-expanding, metallic stents (SEMSs) as a transition to silicone stent implantation for treating severe PTBS. Methods: We retrospectively reviewed the data of patients with severe PTBS who received airway stenting in the First Affiliated Hospital of Guangdong Medical University between September 2015 and May 2019. The types of the stent, intervention procedures, bronchoscopic findings, clinical outcomes and related complications were collected and analyzed. Results: Fifty-eight cases with severe PTBS were included in this study. Thirteen (22.4%) of the patients received bronchial silicone stent implantation immediately after dilations. For the remaining 45 (77.6%) patients, silicone stents could not be deployed after dilations and SEMSs implantation was implemented as a bridge to silicone stenting. The SEMSs were placed for an interval of 28.4 ± 11.1 days. All of the silicone stents were inserted successfully following the removal of SEMSs. No SEMS-related complication occurred. The subgroup analysis showed that patients who received transitional SEMSs had less luminal caliber but fewer transbronchial dilations before silicone stent implantation ( p Conclusion: Covered SEMS placement as a transition to silicone stenting could serve as a feasible procedure to reduce complications and improve the success rate of silicone stent implantation in patients with severe PTBS. The reviews of this paper are available via the supplemental material section.

Most biological functions involve protein&ndash, 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&prime, s IDR undergoes the neurodegenerative disease related &alpha, helix-to-&beta, sheet transition increases in relation to the amount of hnRNP-A2&prime, 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&rsquo, s functions through their IDRs.

Liquid-liquid phase separation (LLPS) explains many intracellular activities, but its role in extracellular functions has not been studied to the same extent. Here we report how LLPS mediates the extracellular function of galectin-3, the only monomeric member of the galectin family. The mechanism through which galectin-3 agglutinates (acting as a “bridge” to aggregate glycosylated molecules) is largely unknown. Our data show that its N-terminal domain (NTD) undergoes LLPS driven by interactions between its aromatic residues (two tryptophans and 10 tyrosines). Our lipopolysaccharide (LPS) micelle model shows that the NTDs form multiple weak interactions to other galectin-3 and then aggregate LPS micelles. Aggregation is reversed when interactions between the LPS and the carbohydrate recognition domains are blocked by lactose. The proposed mechanism explains many of galectin-3’s functions and suggests that the aromatic residues in the NTD are interesting drug design targets., Galectin-3 consists of an unstructured N-terminal domain (NTD) and a structured carbohydrate-recognition domain and agglutinates neutrophils and glycosylated molecules in the extracellular milieu. Here the authors combine biophysical and biochemical experiments with NMR measurements and show that the galectin-3 NTD undergoes liquid-liquid phase separation (LLPS) and agglutinates other molecules through this process.

Eukaryotic cells contain distinct organelles, but not all of these compartments are enclosed by membranes. Some intrinsically disordered proteins mediate membraneless organelle formation through liquid–liquid phase separation (LLPS). LLPS facilitates many biological functions such as regulating RNA stability and ribonucleoprotein assembly, and disruption of LLPS pathways has been implicated in several diseases. Proteins exhibiting LLPS typically have low sequence complexity and specific repeat motifs. These motifs promote multivalent connections with other molecules and the formation of higher-order oligomers, and their removal usually prevents LLPS. The intrinsically disordered C-terminal domain of TAR DNA-binding protein 43 (TDP-43), a protein involved in motor neuron disease and dementia lacks a dominant LLPS motif, however, and how this domain forms condensates is unclear. Using extensive mutagenesis of TDP-43, we demonstrate here that three tryptophan residues and, to a lesser extent, four other aromatic residues are most important for TDP-43 to undergo LLPS. Our results also suggested that only a few residues may be required for TDP-43 LLPS because the α-helical segment (spanning ∼20 residues) in the middle part of the C-terminal domain tends to self-assemble, reducing the number of motifs required for forming a multivalent connection. Our results indicating that a self-associating α-helical element with a few key residues regulates condensate formation highlight a different type of LLPS involving intrinsically disordered regions. The C-terminal domain of TDP-43 contains ∼50 disease-related mutations, with no clear physicochemical link between them. We propose that they may disrupt LLPS indirectly by interfering with the key residues identified here.

The TAR DNA-binding protein of 43kDa (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.

Galectins are a family of lectins that bind β-galactosides through their conserved carbohydrate recognition domain (CRD) and can induce aggregation with glycoproteins or glycolipids on the cell surface and thereby regulate cell activation, migration, adhesion, and signaling. Galectin-3 has an intrinsically disordered N-terminal domain and a canonical CRD. Unlike the other 14 known galectins in mammalian cells, which have dimeric or tandem-repeated CRDs enabling multivalency for various functions, galectin-3 is monomeric, and its functional multivalency therefore is somewhat of a mystery. Here, we used NMR spectroscopy, mutagenesis, small-angle X-ray scattering, and computational modeling to study the self-association–related multivalency of galectin-3 at the residue-specific level. We show that the disordered N-terminal domain (residues ∼20–100) interacts with itself and with a part of the CRD not involved in carbohydrate recognition (β-strands 7–9; residues ∼200–220), forming a fuzzy complex via inter- and intramolecular interactions, mainly through hydrophobicity. These fuzzy interactions are characteristic of intrinsically disordered proteins to achieve liquid–liquid phase separation, and we demonstrated that galectin-3 can also undergo liquid–liquid phase separation. We propose that galectin-3 may achieve multivalency through this multisite self-association mechanism facilitated by fuzzy interactions.

// Cheng-Te Hsiao 1, 2, * , Hung-Wei Cheng 3, * , Chi-Ming Huang 3 , Hao-Ru Li 3 , Meng-Hsin Ou 3 , Jie-Rong Huang 3 , Kay-Hooi Khoo 1, 2 , Helen Wenshin Yu 4 , Yin-Quan Chen 4 , Yang-Kao Wang 5 , Arthur Chiou 4, 6 and Jean-Cheng Kuo 3, 4, 7 1 Institute of Biochemical Sciences, National Taiwan University, Taipei 10617, Taiwan 2 Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan 3 Institute of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei 11221, Taiwan 4 Biophotonics and Molecular Imaging Research Center, National Yang-Ming University, Taipei 11221, Taiwan 5 Department of Cell Biology and Anatomy, National Cheng Kung University, Tainan 70101, Taiwan 6 Institute of Biophotonics, National Yang-Ming University, Taipei 11221, Taiwan 7 Proteomics Research Center, National Yang-Ming University, Taipei 11221, Taiwan * These authors have contributed equally to this work Correspondence to: Jean-Cheng Kuo, email: jckuo@ym.edu.tw Keywords: fibronectin, glycoproteomics, N-glycans, integrin signals, cell migration Received: November 04, 2016 Accepted: July 18, 2017 Published: August 07, 2017 ABSTRACT Directed cell migration is an important step in effective wound healing and requires the dynamic control of the formation of cell-extracellular matrix interactions. Plasma fibronectin is an extracellular matrix glycoprotein present in blood plasma that plays crucial roles in modulating cellular adhesion and migration and thereby helping to mediate all steps of wound healing. In order to seek safe sources of plasma fibronectin for its practical use in wound dressing, we isolated fibronectin from human (homo) and porcine plasma and demonstrated that both have a similar ability as a suitable substrate for the stimulation of cell adhesion and for directing cell migration. In addition, we also defined the N-glycosylation sites and N-glycans present on homo and porcine plasma fibronectin. These N-glycosylation modifications of the plasma fibronectin synergistically support the integrin-mediated signals to bring about mediating cellular adhesion and directed cell migration. This study not only determines the important function of N-glycans in both homo and porcine plasma fibronectin-mediated cell adhesion and directed cell migration, but also reveals the potential applications of porcine plasma fibronectin if it was applied as a material for clinical wound healing and tissue repair.

The properties of unfolded proteins are essential both for the mechanisms of protein folding and for the function of the large group of intrinsically disordered proteins. However, the detailed structural and dynamical characterization of these highly dynamic and conformationally heterogeneous ensembles has remained challenging. Here we combine and compare three of the leading techniques for the investigation of unfolded proteins, NMR spectroscopy (NMR), small-angle X-ray scattering (SAXS), and single-molecule Förster resonance energy transfer (FRET), with the goal of quantitatively testing their consistency and complementarity and for obtaining a comprehensive view of the unfolded-state ensemble. Using unfolded ubiquitin as a test case, we find that its average dimensions derived from FRET and from structural ensembles calculated using the program X-PLOR-NIH based on NMR and SAXS restraints agree remarkably well; even the shapes of the underlying intramolecular distance distributions are in good agreement, attesting to the reliability of the approaches. The NMR-based results provide a highly sensitive way of quantifying residual structure in the unfolded state. FRET-based nanosecond fluorescence correlation spectroscopy allows long-range distances and chain dynamics to be probed in a time range inaccessible by NMR. The combined techniques thus provide a way of optimally using the complementarity of the available methods for a quantitative structural and dynamical description of unfolded proteins both at the global and the local level.

In NMR experiments, the chemical shift is typically the first parameter measured and is a source of structural information for biomolecules. Indeed, secondary chemical shifts, the difference between the measured chemical shifts and those expected for a randomly oriented sequence of peptides (theamp;quot;random coilamp;quot;), are correlated with the secondary structure of proteins; secondary shift analysis is thereby a standard approach in structural biology. For intrinsically disordered or denatured proteins furthermore, secondary chemical shifts reveal the propensity of particular segments to form different secondary structures. However, because the atoms in unfolded proteins all have very similar chemical environments, the chemical shifts measured for a certain atom type vary less than in globular proteins. Since chemical shifts can be measured precisely, the secondary chemical shifts calculated for an unfolded system depend mainly on the particular random coil chemical shift database chosen as a point of reference. Certain databases correct the random coil shift for a given residue based on its neighbors in the amino acid sequence. However, these corrections are typically derived from the analysis of model peptides; there have been relatively few direct and systematic studies of the effect of neighboring residues for specific amino acid sequences in disordered proteins. For the study reported here, we used the intrinsically disordered C-terminal domain of TDP-43, which has a highly repetitive amino-acid sequence, as a model system. We assigned the chemical shifts of this protein at low pH in urea. Our results demonstrate that the identity of the nearest neighbors is decisive in determining the value of the chemical shift for atoms in a random coil arrangement. Based on these observations, we also outline a possible approach to construct a random-coil library of chemical shifts that comprises all possible arrangement of tripeptides from a manageable number of polypeptides.

International audience; 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.

Many functional proteins do not have well defined folded structures. In recent years, both experimental and computational approaches have been developed to study the conformational behaviour of this type of protein. It has been shown previously that experimental RDCs (residual dipolar couplings) can be used to study the backbone sampling of disordered proteins in some detail. In these studies, the backbone structure was modelled using a common geometry for all amino acids. In the present paper, we demonstrate that experimental RDCs are also sensitive to the specific geometry of each amino acid as defined by energy-minimized internal co-ordinates. We have modified the FM (flexible-Meccano) algorithm that constructs conformational ensembles on the basis of a statistical coil model, to account for these differences. The modified algorithm inherits the advantages of the FM algorithm to efficiently sample the potential energy landscape for coil conformations. The specific geometries incorporated in the new algorithm result in a better reproduction of experimental RDCs and are generally applicable for further studies to characterize the conformational properties of intrinsically disordered proteins. In addition, the internal-co-ordinate-based algorithm is an order of magnitude more efficient, and facilitates side-chain construction, surface osmolyte simulation, spin-label distribution sampling and proline cis/trans isomer simulation.

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

The molecular details of how urea interacts with, and eventually denatures proteins, remain largely unknown. In this study we have used extensive experimental NMR data, in combination with statistical coil ensemble modeling and small-angle scattering, to analyze the conformational behavior of the protein ubiquitin in the presence of urea. In order to develop an atomic resolution understanding of the denatured state, conformational ensembles of full-atom descriptions of unfolded proteins, including side chain conformations derived from rotamer libraries, are combined with random sampling of explicit urea molecules in interaction with the protein. Using this description of the conformational equilibrium, we demonstrate that the direct-binding model of urea to the protein backbone is compatible with available experimental data. We find that, in the presence of 8 M urea, between 30 and 40% of the backbone peptide groups bind a urea molecule, independently reproducing results from a model-free analysis of small-angle neutron and X-ray scattering data. Crucially, this analysis also provides sequence specific details of the interaction between urea and the protein backbone. The pattern of urea-binding along the amino-acid sequence reveals a higher level of binding in the central part of the protein, a trend which resembles independent results derived from chemical shift mapping of the urea-protein interaction. Together these results substantiate the direct-binding model and provide a framework for studying the physical basis of interactions between proteins and solvent molecules.

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 (3)J(HalphaHbeta), (3)J(NHbeta), and (3)J(C'Hbeta) scalar and (1)D(CbetaHbeta) residual dipolar couplings (RDCs) in unfolded proteins. For urea-denatured ubiquitin and protein G, up to six (3)J-couplings to (1)H(beta) are detected, which define the chi(1) angle at very high precision. Interpretation of the (3)J couplings by a model of mixed staggered chi(1) rotamers yields excellent agreement and also provides stereoassignments for (1)H(beta) methylene protons. For all observed amino acids with the exception of leucine, the chemical shift of (1)H(beta3) protons was found downfield from (1)H(beta2). For most residues, the precision of individual chi(1) rotamer populations is better than 2%. The experimental chi(1) 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 (1)D(CbetaHbeta) RDCs correlate well with predicted RDCs that were calculated from a sterically aligned coil model ensemble and the (3)J-derived chi(1) 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.

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 Delta G(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 beta-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 beta-strands 7, 8, 9 and 10 have, on average, higher exchange rates than others in the beta-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 beta-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.

Green fluorescent protein (GFP) and its many variants are probably the most widely used proteins in medical and biological research, having been extensively engineered to act as markers of gene expression and protein localization, indicators of protein-protein interactions and biosensors. GFP first folds, before it can undergo an autocatalytic cyclization and oxidation reaction to form the chromophore, and in many applications the folding efficiency of GFP is known to limit its use. Here, we review the recent literature on protein engineering studies that have improved the folding properties of GFP. In addition, we discuss in detail the biophysical work on the folding of GFP that is beginning to reveal how this large and complex structure forms.

International audience; Multidomain proteins containing intrinsically disordered linkers exhibit large-scale dynamic modes that play key roles in a multitude of molecular recognition and signaling processes. Here, we determine the conformational space sampled by the multidomain splicing factor U2AF65 using complementary nuclear magnetic resonance spectroscopy and small-angle scattering data. Available degrees of conformational freedom are initially stochastically sampled and experimental data then used to delineate the potential energy landscape in terms of statistical probability. The spatial distribution of U2AF65 conformations is found to be highly anisotropic, comprising significantly populated interdomain contacts that appear to be electrostatic in origin. This hypothesis is supported by the reduction of signature PREs reporting on expected interfaces with increasing salt concentration. The described spatial distribution reveals the complete spectrum of the unbound forms of U2AF65 that coexist with the small percentage of a preformed RNA-bound domain arrangement required for polypyrimidine-tract recognition by conformational selection. More generally, the proposed approach to describing conformational equilibria of multidomain proteins can be further combined with other experimental data that are sensitive to domain dynamics.

International audience

Intrinsically disordered regions are predicted to exist in a significant fraction of proteins encoded in eukaryotic genomes. The high levels of conformational plasticity of this class of proteins endows them with unique capacities to act in functional modes not achievable by folded proteins, but also places their molecular characterization beyond the reach of classical structural biology. New techniques are therefore required to understand the relationship between primary sequence and biological function in this class of proteins. Although dependences of some NMR parameters such as chemical shifts (CSs) or residual dipolar couplings (RDCs) on structural propensity are known, so that sampling regimes are often inferred from experimental observation, there is currently no framework that allows for a statistical mapping of the available Ramachandran space of each amino acid in terms of conformational propensity. In this study we develop such an approach, combining highly efficient conformational sampling with ensemble selection to map the backbone conformational sampling of IDPs on a residue specific level. By systematically analyzing the ability of NMR data to map the conformational landscape of disordered proteins, we identify combinations of RDCs and CSs that can be used to raise conformational degeneracies inherent to different data types, and apply these approaches to characterize the conformational behavior of two intrinsically disordered proteins, the K18 domain from Tau protein and N(TAIL) from measles virus nucleoprotein. In both cases, we identify the enhanced populations of turn and helical regions in key regions of the proteins, as well as contiguous strands that show clear and enhanced polyproline II sampling.

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.

In order to understand the conformational behaviour of Intrinsically Disordered Proteins (IDPs), it is essential to develop a molecular representation of the partially folded state. Due to the very large number of degrees of conformational freedom available to such a disordered system, this problem is highly underdetermined. Characterisation therefore requires extensive experimental data, and novel analytical tools are required to exploit the specific conformational sensitivity of different experimental parameters. In this review we concentrate on the use of nuclear magnetic resonance (NMR) spectroscopy for the study of conformational behaviour of IDPs at atomic resolution. Each experimental NMR parameter is sensitive to different aspects of the structural and dynamic behaviour of the disordered state and requires specific consideration of the relevant averaging properties of the physical interaction. In this review we present recent advances in the description of disordered proteins and the selection of representative ensembles on the basis of experimental data using statistical coil sampling from flexible-meccano and ensemble selection using ASTEROIDS. Using these tools we aim to develop a unified molecular representation of the disordered state, combining complementary data sets to extract a meaningful description of the conformational behaviour of the protein.

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. C(alpha) 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 beta-hairpin and alpha-helix as well as non-native alpha-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 C(alpha) 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.

A new lanthanide chelating tag (M8) for paramagnetic labeling of biomolecules is presented, which is based on an eight-fold, stereoselectively methyl-substituted DOTA that can be covalently linked to the host molecule by a single disulfide bond. The steric overcrowding of the DOTA scaffold leads to an extremely rigid, kinetically and chemically inert lanthanide chelator. Its steric bulk restricts the motion of the tag relative to the host molecule. These properties result in very large pseudocontact shifts (>5 ppm) and residual dipolar couplings (>20 Hz) for Dy-M8 linked to ubiquitin, which are unprecedented for a small, single-point-attachment tag. Such large pseudocontact shifts should be well detectable even for larger proteins and distances beyond approximately 50 A. Due to its exceptionally high stability and lanthanide affinity M8 can be used under extreme chemical or physical conditions, such as those applied for protein denaturation, or when it is undesirable that buffer or protein react with excess lanthanide ions.

Green fluorescent protein „GFP… is a large protein with a complex elevenstranded -barrel structure. Previous studies have shown that it has a complex energy landscape for folding on which there are several intermediate states and a denatured state with significant residual structure. Here, we use two different types of H/D exchange measurement and nuclear magnetic resonance „NMR… techniques to probe the energy landscape for folding of GFP in further detail. H/D exchange experiments were performed over a wide range of conditions including different concentrations of denaturant. Results show that the penetration model dominates the exchange mechanism, consistent with the known stability and slow unfolding kinetics of GFP. H/D exchange experiments at high pH establish that there is an extremely slow-exchanging superstable core of amide protons in GFP that are clustered and located in -strands 1, 2, 4, 5, and 6. These residues form part of a mini--sheet which we propose constitutes a folding nucleus. Using a pulsed-labeling strategy, the acid-denatured state has been investigated and the residual structure observed in earlier studies shown to locate to -strands 1 and 3. There is some evidence that this residual structure is stabilized by a localized hydrophobic collapse of the polypeptide chain. [DOI: 10.2976/1.2976660]