Coil State Research Articles

Effects of Charge Interactions and Transient Secondary Structure Elements on the Function of the Disordered RAM Region of the Notch Receptor

The Notch signaling pathway is crucial for cellular development. The intracellular domain of the Notch receptor (NICD) contains the 140-residue intrinsically disordered RAM region followed by seven ankyrin repeats, a nuclear localization sequence, and a C-terminal degradation sequence. The RAM N-terminus and the ankyrin repeats of NICD form a bivalent interaction with the transcription factor CSL that leads to the activation of Notch target genes. Assays measuring transcription activity of NICD variants containing sequence deletions and insertions in the region of RAM between the N-terminal binding site and the ankyrin repeats reveal that RAM mostly behaves as a disordered statistical coil. However, deviations from this model suggest that NICD bivalency is affected by sequence-specific elements within RAM, such as transient secondary structure elements and/or charge interactions. NMR spectroscopy has revealed three regions of RAM with helical propensity and two regions that are extended and dynamic. Transcription assays show that sequence substitutions to these regions have a modest effect on transcriptional activation. In contrast, charge interactions within the RAM are crucial for transcriptional activation. Redistributing the charged residues within RAM changes the tertiary structure of RAM and decreases transcriptional activation. NICD variants with highly segregated positive and negative charges in the RAM region are more compact than wild-type RAM and display almost no transcriptional activation. Variants containing oppositely charged residues that are more mixed than in wild-type RAM are more extended and exhibit smaller decreases in transcriptional activation. Current work is focused on understanding why compaction of the RAM region affects Notch transcriptional activation. Understanding how the secondary and tertiary structure elements of RAM affect transcriptional activation will provide insight into the general roles of IDPs in signaling pathways.

Intrinsic disorder within the erythrocyte binding-like proteins from Plasmodium falciparum

The ability of the malaria parasite, Plasmodium falciparum, to proliferate within the human host depends on its invasion of erythrocytes. Erythrocyte binding-like (EBL) proteins play crucial roles in the attachment of merozoites to human erythrocytes by binding to specific receptors on the cell surface. In this study, we have carried out a bioinformatics analysis of the three EBL proteins EBA-140, EBA-175 and EBA-181 and show that they contain a large amount of intrinsic disorder in particular within the RIII–V domains. The functional role of these domains has so far not been identified, although antibodies raised against these regions were shown to inhibit parasite invasion. Here, we obtain a more complete structural and dynamic view of the EBL proteins by focusing on the biophysical characterization of a smaller construct of the RIII–V regions of EBA-181 (EBA-181945–1097). We show using a number of techniques that EBA-181945–1097 is intrinsically disordered, and we obtain a detailed structural and dynamic characterization of the protein at atomic resolution using nuclear magnetic resonance (NMR) spectroscopy. Our results show that EBA-181945–1097 is essentially a statistical coil with the presence of several turn motifs and does not possess transiently populated secondary structures as is common for many intrinsically disordered proteins that fold via specific, pre-formed molecular recognition elements.

Finite-Size Conformational Transitions: A Unifying Concept Underlying Chromosome Dynamics

Investigating average thermodynamic quantities is not sufficient to understand conformational transitions of a finite-size polymer. We propose that such transitions are better described in terms of the probability distribution of some finite-size order parameter, and the evolution of this distribution as a control parameter varies. We demonstrate this claim for the coil-globule transition of a linear polymer and its mapping onto a two-state model. In a biological context, polymer models delineate the physical constraints experienced by the genome at different levels of organization, from DNA to chromatin to chromosome. We apply our finite-size approach to the formation of plectonemes in a DNA segment submitted to an applied torque and the ensuing helix-coil transition that can be numerically observed, with a coexistence of the helix and coil states in a range of parameters. Polymer models are also essential to analyze recent in vivo experiments providing the frequency of pairwise contacts between genomic loci. The probability distribution of these contacts yields quantitative information on the conformational fluctuations of chromosome regions. The changes observed in the shape of the distribution when the cell type or the physiological conditions vary may reveal an epigenetic modulation of the conformational constraints experienced by the chromosomes.

Loading capacity and interaction of DNA binding on catanionic vesicles with different cationic surfactants.

Cationic and anionic (catanionic) vesicles were constructed from the mixtures of sodium laurate (SL) and alkyltrimethylammonium bromide (CnTAB, n = 12, 14, and 16) and were used to control the loading capacity of DNA. The binding saturation point (BSP) of DNA to catanionic vesicles increases with the chain length of cationic surfactants, which is at 1.0, 1.3 and 1.5 for CnTAB with n = 12, 14, and 16, respectively. Our measurements showed that the loading capacity and affinity of DNA can be controlled by catanionic vesicles. It increases with the chain length of cationic surfactants. Because of a large reduction in surface charge density, catanionic vesicles are prone to undergo re-aggregation or fusion with the addition of DNA. DNA molecules can still maintain original coil state during the interaction with catanionic CnTAL vesicles. (1)H NMR data reveals that the obvious dissociation of anionic ions, L(-), from catanionic C14TAL vesicles is due to the interaction with DNA; however, this phenomenon cannot be observed in C12TAB-SL vesicles. Agarose gel electrophoresis (AGE) results demonstrate that the electrostatic interaction between the two oppositely charged cationic and anionic surfactants is stronger than that between DNA and cationic surfactant, CnTAB (n = 12, 14, and 16). Not only is the dissociation of L(-) simply determined by the charge competition, but it also depends largely on the variations in the surface charge density as well as the cationic and anionic surfactant competing ability in geometry configuration of catanionic vesicles. The complicated interaction between DNA and catanionic vesicles induces the deformation of cationic vesicles. Our results should provide clear guidance for choosing more proper vectors for DNA delivery and gene therapy in cell experiments.

Induction of Peptide Bond Dipoles Drives Cooperative Helix Formation in the (AAQAA)3 Peptide

Cooperativity is a central feature in the formation of secondary structures in proteins. However, the driving forces behind this cooperativity are poorly understood. The present work shows that the cooperativity of helix formation in the acetyl-(AAQAA)3-NH2 peptide is significantly enhanced using an empirical force field that explicitly includes the treatment of electronic polarizability. Polarizable simulations yield helical content consistent with experimental measurements and indicate that the dependence of helical content on temperature is improved over additive models, though further sampling is required to fully validate this conclusion. Cooperativity is indicated by the peptide sampling either the coiled state or long helices with relatively low populations of short helices. The cooperativity is shown to be associated with enhanced dipole moments of the peptide backbone upon helix formation. These results indicate the polarizable force field to more accurately model peptide-folding cooperativity based on its physically realistic treatment of electronic polarizability.

Coil-bridge transition in a single polymer chain as an unconventional phase transition: theory and simulation.

The coil-bridge transition in a self-avoiding lattice chain with one end fixed at height H above the attractive planar surface is investigated by theory and Monte Carlo simulation. We focus on the details of the first-order phase transition between the coil state at large height H ⩾ Htr and a bridge state at H ⩽ Htr, where Htr corresponds to the coil-bridge transition point. The equilibrium properties of the chain were calculated using the Monte Carlo pruned-enriched Rosenbluth method in the moderate adsorption regime at (H/Na)tr ⩽ 0.27 where N is the number of monomer units of linear size a. An analytical theory of the coil-bridge transition for lattice chains with excluded volume interactions is presented in this regime. The theory provides an excellent quantitative description of numerical results at all heights, 10 ⩽ H/a ⩽ 320 and all chain lengths 40 < N < 2560 without free fitting parameters. A simple theory taking into account the effect of finite extensibility of the lattice chain in the strong adsorption regime at (H/Na)tr ⩾ 0.5 is presented. We discuss some unconventional properties of the coil-bridge transition: the absence of phase coexistence, two micro-phases involved in the bridge state, and abnormal behavior in the microcanonical ensemble.

Molecular-level changes of aqueous poly(N-isopropylacrylamide) in phase transition.

We report a Compton scattering study on the molecular-level structural changes of aqueous poly(N-isopropylacrylamide) (PNIPAM) across the conformational phase transition. PNIPAM is a thermoresponsive polymer that changes its conformation in water from the hydrophilic coil state to the collapsed hydrophobic globule state at 32 °C. Combined with density functional theory calculations, the Compton scattering experiments detect two type of changes in the phase transition. The amount of hydrogen bonds is found to reduce, and an elongation of the internal covalent bond lengths is observed. The elongation of the bonds indicates that not only the hydrogen bonding changes but there are other processes, most likely related to hydrophobic interaction, that should be taken into account in the phase transition.

Unravelling Secondary Structure Changes on Individual Anionic Polysaccharide Chains by Atomic Force Microscopy

AbstractThe structural conformations of the anionic carrageenan polysaccharides in the presence of monovalent salt close to physiological conditions are studied by atomic force microscopy. Iota‐carrageenan undergoes a coil–helix transition at high ionic strength, whereas lambda‐carrageenan remains in the coiled state. Polymer statistical analysis reveals an increase in persistence length from 22.6±0.2 nm in the random coil, to 26.4±0.2 nm in the ordered helical conformation, indicating an increased rigidity of the helical iota‐carrageenan chains. The many decades‐long debated issue on whether the ordered state can exist as single or double helix, is conclusively resolved by demonstrating the existence of a unimeric helix formed intramolecularly by a single polymer chain.

Unravelling secondary structure changes on individual anionic polysaccharide chains by atomic force microscopy.

The structural conformations of the anionic carrageenan polysaccharides in the presence of monovalent salt close to physiological conditions are studied by atomic force microscopy. Iota-carrageenan undergoes a coil-helix transition at high ionic strength, whereas lambda-carrageenan remains in the coiled state. Polymer statistical analysis reveals an increase in persistence length from 22.6±0.2 nm in the random coil, to 26.4±0.2 nm in the ordered helical conformation, indicating an increased rigidity of the helical iota-carrageenan chains. The many decades-long debated issue on whether the ordered state can exist as single or double helix, is conclusively resolved by demonstrating the existence of a unimeric helix formed intramolecularly by a single polymer chain.

The viscometric behaviour of sodium hyaluronate in aqueous and KCl solutions

This paper deals with the viscometric characterization of sodium hyaluronate aqueous and (1:1) electrolyte (KCl) solutions at defined pH and temperature. The solution properties affected the macromolecular chain coil dimensions by triggering their expansion or contraction. Studied hyaluronate solutions viscosities at temperature range of 25–35°C reflect conformation transition from a double helix to statistical coil as revealed by a step change of Huggin’ constant at 30°C. A change in the slope of the observed reduced specific viscosity induced by temperature change was a further indication of the double helix-coil transition. The effects were simultaneously confirmed by fluorescence spectrometry measurements.

Reversible DNA Compaction

In this review we summarize and discuss the different methods we can use to achieve reversible DNA compaction in vitro. Reversible DNA compaction is a natural process that occurs in living cells and viruses. As a result these process long sequences of DNA can be concentrated in a small volume (compacted) to be decompacted only when the information carried by the DNA is needed. In the current work we review the main artificial compacting agents looking at their suitability for decompaction. The different approaches used for decompaction are strongly influenced by the nature of the compacting agent that determines the mechanism of compaction. We focus our discussion on two main artificial compacting agents: multivalent cations and cationic surfactants that are the best known compacting agents. The reversibility of the process can be achieved by adding chemicals like divalent cations, alcohols, anionic surfactants, cyclodextrins or by changing the chemical nature of the compacting agents via pH modifications, light induced conformation changes or by redox-reactions. We stress the relevance of electrostatic interactions and self-assembly as a main approach in order to tune up the DNA conformation in order to create an on-off switch allowing a transition between coil and compact states. The recent advances to control DNA conformation in vitro, by means of molecular self-assembly, result in a better understanding of the fundamental aspects involved in the DNA behavior in vivo and serve of invaluable inspiration for the development of potential biomedical applications.

Conformational changes of DNA in the presence of 12-s-12 gemini surfactants (s = 2 and 10). Role of the spacer's length in the interaction surfactant-polynucleotide

A multifaceted study on the interaction of calf-thymus DNA with two different cationic gemini surfactants alkanediyl-α-ω-bis(dodecyldimethyl-amonium)bromide, 12-s-12,2Br− (with s=2, G2, and 10, G10) was carried out. The measurements were done at different molar ratios X=[surfactant]/[DNA]. Results show two different conformational changes in DNA: a first compaction of the polynucleotide corresponding to a partial conformational (not total) change of DNA from an extended coil state to a globular state that happens at the lower molar ratio X. A second change corresponds to a breaking of the partial condensation, that is, the transition from the compacted state to a new more extended conformation (for the higher X values) different to the initial extension. According to circular dichroism spectra and dynamic light scattering measurements, this new state of DNA seems to be similar to a ψ-phase. Measurements confirm that interactions involved in the compaction are different to those previously obtained for the analog surfactant CTAB. X values at which the conformational changes happen depend on the length of the spacer in the surfactant along with the charge of the polar heads.

Conformational transition of H-shaped branched polymers

We report dynamic Monte Carlo simulation on conformational transition of H-shaped branched polymers by varying main chain (backbone) and side chain (branch) length. H-shaped polymers in comparison with equivalent linear polymers exhibit a depression of theta temperature accompanying with smaller chain dimensions. We observed that the effect of branches on backbone dimension is more pronounced than the reverse, and is attributed to the conformational heterogeneity prevails within the molecule. With an increase in branch length, backbone is slightly stretched out in the coil and globule state. However, in the pre-collapsed (cf. crumpled globule) state, backbone size decreases with the increase of branch length. We attribute this non-monotonic behavior as the interplay between excluded volume interaction and intra-chain bead-bead attractive interaction during collapse transition. Structural analysis reveals that the inherent conformational heterogeneity promotes the formation of a collapsed structure with segregated backbone and branch units (resembles to "sandwich" or "Janus" morphology) rather an evenly distributed structure consisting of all the units. The shape of the collapsed globule becomes more spherical with increasing either backbone or branch length.

Predicting the Predictive Power of IDP Ensembles

The function of intrinsically disordered proteins may be interpreted in terms of their structural ensembles. The article by Schwalbe and colleagues in this issue of Structure combines NMR and SAXS constraints to generate structural ensembles that unveil important functional and pathological features.

P15PAF Is an Intrinsically Disordered Protein with Nonrandom Structural Preferences at Sites of Interaction with Other Proteins

We present to our knowledge the first structural characterization of the proliferating-cell-nuclear-antigen-associated factor p15PAF, showing that it is monomeric and intrinsically disordered in solution but has nonrandom conformational preferences at sites of protein-protein interactions. p15PAF is a 12 kDa nuclear protein that acts as a regulator of DNA repair during DNA replication. The p15PAF gene is overexpressed in several types of human cancer. The nearly complete NMR backbone assignment of p15PAF allowed us to measure 86 N-HN residual dipolar couplings. Our residual dipolar coupling analysis reveals nonrandom conformational preferences in distinct regions, including the proliferating-cell-nuclear-antigen-interacting protein motif (PIP-box) and the KEN-box (recognized by the ubiquitin ligase that targets p15PAF for degradation). In accordance with these findings, analysis of the 15N R2 relaxation rates shows a relatively reduced mobility for the residues in these regions. The agreement between the experimental small angle x-ray scattering curve of p15PAF and that computed from a statistical coil ensemble corrected for the presence of local secondary structural elements further validates our structural model for p15PAF. The coincidence of these transiently structured regions with protein-protein interaction and posttranslational modification sites suggests a possible role for these structures as molecular recognition elements for p15PAF.

High Pressure Effect on a Helical Peptide Studied by Simulated Tempering Molecular Dynamics Simulations

High pressure effect on proteins and peptides has recently been studied theoretically and experimentally to understand structural properties of proteins such as protein folding and the function of proteins. While some proteins are denatured under high pressure conditions, some peptides are more stable at high pressure than at atmospheric pressure. It is important to understand the molecular mechanism of pressure-induced structural changes of proteins and peptides.We studied pressure effect on the structure of a helical peptide by using molecular dynamics simulations. We used an AK16 peptide as a helical peptide. AK16 is more helical under high pressure conditions than at atmospheric pressure. Molecular simulations often get trapped in the local minimum states of the free energy. In order to overcome such difficulty, we used a generalized-ensemble algorithm, which gives more efficient sampling in a molecular simulation. The simulated tempering method for the isobaric-isothermal ensemble (Y. Mori and Y. Okamoto, JPSJ 79, 074003, 2010) was used for efficient sampling in pressure space.We performed molecular dynamics simulations with the simulated tempering method for the system of AK16 in explicit water molecules. We found that the population of the helical structure of the peptide increases as pressure increases. We also calculated the free energy difference and the partial molar volume change as a function of pressure. The partial molar volume change from the helical state to the coil state increased monotonically from a negative value to a positive value with pressure. The positive value of the partial molar volume change is consistent with experimental results. We revealed the molecular details of AK16 under high pressure conditions (Y. Mori and H. Okumura, JPCL 4, 2079, 2013).

Chemical structural features and anti-complementary activity of polysaccharide HPS1-D from Hedysarum polybotrys

HPS1-D, an active polysaccharide,was isolated and purified from Hedysarum polybotrys. HPS1-D was obtained after treated with Savage method and H2O2, and purified with DEAE-cellulose 52 and Sephadex G-100 gel filtration chromatography. Then physicochemical property analysis, GC, methylation, partial acid hydrolysis, and NMR method were used to study chemical structural of HPS1-D. The conformation was primarily analyzed with GPC-MALLS method and Congo red reaction. The anti-complementary activity of HPS1-D was evaluated with the hemolysis assay. HPS1-D was a heteropolysaccharide and consisted of D-glucose, L-arabinose, (7.2:1.3). HPS1-D proved to be a neutral sugar, with 1, 4-and 1, 4, 6-alpha-D-glucopyranosyl residues in backbone ,and 1, 5-and 1, 3, 5-alpha-L-arabinofuranosyl residues in branches. HPS1-D has a random coil state conformation with monodisperse mass distribution in 0.9% NaCl solution. And HPS1-D had triple-helix conformation in concentrate of NaOH solution. Anti-complementary activity of HPS1-D was closed to its positive control heparin.

Predictive Atomic Resolution Descriptions of Intrinsically Disordered hTau40 and α-Synuclein in Solution from NMR and Small Angle Scattering

The development of molecular descriptions of intrinsically disordered proteins (IDPs) is essential for elucidating conformational transitions that characterize common neurodegenerative disorders. We use nuclear magnetic resonance, small angle scattering, and molecular ensemble approaches to characterize the IDPs Tau and α-synuclein. Ensemble descriptions of IDPs are highly underdetermined due to the inherently large number of degrees of conformational freedom compared with available experimental measurements. Using extensive cross-validation we show that five different types of independent experimental parameters are predicted more accurately by selected ensembles than by statistical coil descriptions. The improvement increases in regions whose local sampling deviates from statistical coil, validating the derived conformational description. Using these approaches we identify enhanced polyproline II sampling in aggregation-nucleation sites, supporting suggestions that this region of conformational space is important for aggregation.

Folding thermodynamics of the hybrid‐1 type intramolecular human telomeric G‐quadruplex

Guanine-rich DNA sequences that may form G-quadruplexes are located in strategic DNA loci with the ability to regulate biological events. G-quadruplexes have been under intensive scrutiny owing to their potential to serve as novel drug targets in emerging anticancer strategies. Thermodynamic characterization of G-quadruplexes is an important and necessary step in developing predictive algorithms for evaluating the conformational preferences of G-rich sequences in the presence or the absence of their complementary C-rich strands. We use a combination of spectroscopic, calorimetric, and volumetric techniques to characterize the folding/unfolding transitions of the 26-meric human telomeric sequence d[A3G3(T2AG3)3A2]. In the presence of K+ ions, the latter adopts the hybrid-1 G-quadruplex conformation, a tightly packed structure with an unusually small number of solvent-exposed atomic groups. The K+-induced folding of the G-quadruplex at room temperature is a slow process that involves significant accumulation of an intermediate at the early stages of the transition. The G-quadruplex state of the oligomeric sequence is characterized by a larger volume and compressibility and a smaller expansibility than the coil state. These results are in qualitative agreement with each other all suggesting significant dehydration to accompany the G-quadruplex formation. Based on our volume data, 432±19 water molecules become released to the bulk upon the G-quadruplex formation. This large number is consistent with a picture in which DNA dehydration is not limited to water molecules in direct contact with the regions that become buried but involves a general decrease in solute-solvent interactions all over the surface of the folded structure.

Transition state and ground state properties of the helix–coil transition in peptides deduced from high-pressure studies

Volume changes associated with protein folding reactions contain valuable information about the folding mechanism and the nature of the transition state. However, meaningful interpretation of such data requires that overall volume changes be deconvoluted into individual contributions from different structural components. Here we focus on one type of structural element, the α-helix, and measure triplet-triplet energy transfer at high pressure to determine volume changes associated with the helix-coil transition. Our results reveal that the volume of a 21-amino-acid alanine-based peptide shrinks upon helix formation. Thus, helices, in contrast with native proteins, become more stable with increasing pressure, explaining the frequently observed helical structures in pressure-unfolded proteins. Both helix folding and unfolding become slower with increasing pressure. The volume changes associated with the addition of a single helical residue to a preexisting helix were obtained by comparing the experimental results with Monte Carlo simulations based on a kinetic linear Ising model. The reaction volume for adding a single residue to a helix is small and negative (-0.23 cm(3) per mol = -0.38 Å(3) per molecule) implying that intrahelical hydrogen bonds have a smaller volume than peptide-water hydrogen bonds. In contrast, the transition state has a larger volume than either the helical or the coil state, with activation volumes of 2.2 cm(3)/mol (3.7 Å(3) per molecule) for adding and 2.4 cm(3)/mol (4.0 Å(3) per molecule) for removing one residue. Thus, addition or removal of a helical residue proceeds through a transitory high-energy state with a large volume, possibly due to the presence of unsatisfied hydrogen bonds, although steric effects may also contribute.