<Superscript>1</Superscript>H, <Superscript>15</Superscript>N, <Superscript>13</Superscript>C resonance assignment of plant dehydrin early response to dehydration 10 (ERD10)

Early response to dehydration 10 protein (ERD10) is an intrinsically disordered protein from Arabidopsis thaliana. The protein is upregulated during stress however its mechanism of action at atomic level is not well understood. In the present work multidimensional NMR methodologies are used in order to facilitate the process of chemical shift assignment. The information provided here supports further NMR spectroscopy experiments aimed at elucidation of ERD10 behaviour during molecular recognition events with other proteins.     

Chaperone activity of ERD10 and ERD14, two disordered stress-related plant proteins

ERD10 and ERD14 (for early response to dehydration) proteins are members of the dehydrin family that accumulate in response to abiotic environmental stresses, such as high salinity, drought, and low temperature, in Arabidopsis (Arabidopsis thaliana). Whereas these proteins protect cells against the consequences of dehydration, the exact mode(s) of their action remains poorly understood. Here, detailed evidence is provided that ERD10 and ERD14 belong to the family of intrinsically disordered proteins, and it is shown in various assays that they act as chaperones in vitro. ERD10 and ERD14 are able to prevent the heat-induced aggregation and/or inactivation of various substrates, such as lysozyme, alcohol dehydrogenase, firefly luciferase, and citrate synthase. It is also demonstrated that ERD10 and ERD14 bind to acidic phospholipid vesicles without significantly affecting membrane fluidity. Membrane binding is strongly influenced by ionic strength. Our results show that these intrinsically disordered proteins have chaperone activity of rather wide substrate specificity and that they interact with phospholipid vesicles through electrostatic forces. We suggest that these findings provide the rationale for the mechanism of how these proteins avert the adverse effects of dehydration stresses.     

Secondary structure and dynamics of an intrinsically unstructured linker domain

The transient secondary structure and dynamics of an intrinsically unstructured linker domain from the 70 kDa subunit of human replication protein A was investigated using solution state NMR. Stable secondary structure, inferred from large secondary chemical shifts, was observed for a segment of the intrinsically unstructured linker domain when it is attached to an N-terminal protein interaction domain. Results from NMR relaxation experiments showed the rotational diffusion for this segment of the intrinsically unstructured linker domain to be correlated with the N-terminal protein interaction domain. When the N-terminal domain is removed, the stable secondary structure is lost and faster rotational diffusion is observed. The large secondary chemical shifts were used to calculate phi and psi dihedral angles and these dihedral angles were used to build a backbone structural model. Restrained molecular dynamics were performed on this new structure using the chemical shift based dihedral angles and a single NOE distance as restraints. In the resulting family of structures a large, solvent exposed loop was observed for the segment of the intrinsically unstructured linker domain that had large secondary chemical shifts.     

Exposure to buffer solution alters tendon hydration and mechanics

A buffer solution is often used to maintain tissue hydration during mechanical testing. The most commonly used buffer solution is a physiological concentration of phosphate buffered saline (PBS); however, PBS increases the tissue’s water content and decreases its tensile stiffness. In addition, solutes from the buffer can diffuse into the tissue and interact with its structure and mechanics. These bathing solution effects can confound the outcome and interpretation of mechanical tests. Potential bathing solution artifacts, including solute diffusion, and their effect on mechanical properties, are not well understood. The objective of this study was to measure the effects of long-term exposure of rat tail tendon fascicles to several concentrations (0.9–25%) of NaCl, sucrose, polyethylene glycol (PEG), and SPEG (NaCl + PEG) solutions on water content, solute diffusion, and mechanical properties. We found that with an increase in solute concentration the apparent water content decreased for all solution types. Solutes diffused into the tissue for NaCl and sucrose, however, no solute diffusion was observed for PEG or SPEG. The mechanical properties changed for both NaCl solutions, in particular after long-term (8 h) incubation the modulus and equilibrium stress decreased compared to short-term (15 min) for 25% NaCl, and the cross sectional area increased for 0.9% NaCl. However, the mechanical properties were unchanged for both PEG and SPEG except for minor alterations in stress relaxation parameters. This study shows that NaCl and sucrose buffer solutions are not suitable for long-term mechanical tests. We therefore propose using PEG or SPEG as alternative buffer solutions that after long-term incubation can maintain tissue hydration without solute diffusion and produce a consistent mechanical response.     

Secondary Structure and Dynamics of an Intrinsically Unstructured Linker Domain

Abstract

The transient secondary structure and dynamics of an intrinsically unstructured linker domain from the 70 kDa subunit of human replication protein A was investigated using solution state NMR. Stable secondary structure, inferred from large secondary chemical shifts, was observed for a segment of the intrinsically unstructured linker domain when it is attached to an N-terminal protein interaction domain. Results from NMR relaxation experiments showed the rotational diffusion for this segment of the intrinsically unstructured linker domain to be correlated with the N-terminal protein interaction domain. When the N-terminal domain is removed, the stable secondary structure is lost and faster rotational diffusion is observed. The large secondary chemical shifts were used to calculate phi and psidihedral angles and these dihedral angles were used to build a backbone structural model. Restrained molecular dynamics were performed on this new structure using the chemical shift based dihedral angles and a single NOE distance as restraints. In the resulting family of structures a large, solvent exposed loop was observed for the segment of the intrinsically unstructured linker domain that had large secondary chemical shifts.     

Disordered plant LEA proteins as molecular chaperones

Plants often respond to abiotic stresses by the increased expression of LEA (late embryogenesis abundant) proteins, so called because they also accompany seed formation. Whereas the cellular function of LEA proteins in mitigating the damage caused by stress is clear, the molecular mechanisms of their action are rather enigmatic. Several models have been developed, based on their putative activities as ion sinks, stabilizers of membrane structure, buffers of hydrate water, antioxidants and/or chaperones. Due to their known structural flexibility, this latter idea has received little experimental attention thus far. Recently, however, it has been suggested that intrinsically disordered proteins (IDPs) may exert chaperone activity by an “entropy transfer” mechanism. In our subsequent study published in the May issue of Plant Physiology, we provided evidence that two group 2 LEA proteins, ERD (early response to dehydration) 10 and 14, are potent molecular chaperones. This observation may have far-reaching implications, as it may explain how LEA proteins of ill-defined structures protect plant cells during dehydration, and it may also lead to the general experimental validation of the entropy transfer model of disordered chaperones.Key words: abiotic stress, dehydration stress, stress tolerance, late embryogenesis abundant protein, chaperone, disordered protein, unstructured protein     

Femtosecond Hydration Map of Intrinsically Disordered α-Synuclein

Protein hydration water plays a fundamentally important role in protein folding, binding, assembly, and function. Little is known about the hydration water in intrinsically disordered proteins that challenge the conventional sequence-structure-function paradigm. Here, by combining experiments and simulations, we show the existence of dynamical heterogeneity of hydration water in an intrinsically disordered presynaptic protein, namely α-synuclein, implicated in Parkinson’s disease. We took advantage of nonoccurrence of cysteine in the sequence and incorporated a number of cysteine residues at the N-terminal segment, the central amyloidogenic nonamyloid-β component (NAC) domain, and the C-terminal end of α-synuclein. We then labeled these cysteine variants using environment-sensitive thiol-active fluorophore and monitored the solvation dynamics using femtosecond time-resolved fluorescence. The site-specific femtosecond time-resolved experiments allowed us to construct the hydration map of α-synuclein. Our results show the presence of three dynamically distinct types of water: bulk, hydration, and confined water. The amyloidogenic NAC domain contains dynamically restrained water molecules that are strikingly different from the water molecules present in the other two domains. Atomistic molecular dynamics simulations revealed longer residence times for water molecules near the NAC domain and supported our experimental observations. Additionally, our simulations allowed us to decipher the molecular origin of the dynamical heterogeneity of water in α-synuclein. These simulations captured the quasi-bound water molecules within the NAC domain originating from a complex interplay between the local chain compaction and the sequence composition. Our findings from this synergistic experimental simulation approach suggest longer trapping of interfacial water molecules near the amyloidogenic hotspot that triggers the pathological conversion into amyloids via chain sequestration, chain desolvation, and entropic liberation of ordered water molecules.     

Multiple Interactions of the Intrinsically Disordered Region between the Helicase and Nuclease Domains of the Archaeal Hef Protein

Hef is an archaeal protein that probably functions mainly in stalled replication fork repair. The presence of an unstructured region was predicted between the two distinct domains of the Hef protein. We analyzed the interdomain region of Thermococcus kodakarensis Hef and demonstrated its disordered structure by CD, NMR, and high speed atomic force microscopy (AFM). To investigate the functions of this intrinsically disordered region (IDR), we screened for proteins interacting with the IDR of Hef by a yeast two-hybrid method, and 10 candidate proteins were obtained. We found that PCNA1 and a RecJ-like protein specifically bind to the IDR in vitro. These results suggested that the Hef protein interacts with several different proteins that work together in the pathways downstream from stalled replication fork repair by converting the IDR structure depending on the partner protein.     

Dynamical coupling of intrinsically disordered proteins and their hydration water: comparison with folded soluble and membrane proteins

Hydration water is vital for various macromolecular biological activities, such as specific ligand recognition, enzyme activity, response to receptor binding, and energy transduction. Without hydration water, proteins would not fold correctly and would lack the conformational flexibility that animates their three-dimensional structures. Motions in globular, soluble proteins are thought to be governed to a certain extent by hydration-water dynamics, yet it is not known whether this relationship holds true for other protein classes in general and whether, in turn, the structural nature of a protein also influences water motions. Here, we provide insight into the coupling between hydration-water dynamics and atomic motions in intrinsically disordered proteins (IDP), a largely unexplored class of proteins that, in contrast to folded proteins, lack a well-defined three-dimensional structure. We investigated the human IDP tau, which is involved in the pathogenic processes accompanying Alzheimer disease. Combining neutron scattering and protein perdeuteration, we found similar atomic mean-square displacements over a large temperature range for the tau protein and its hydration water, indicating intimate coupling between them. This is in contrast to the behavior of folded proteins of similar molecular weight, such as the globular, soluble maltose-binding protein and the membrane protein bacteriorhodopsin, which display moderate to weak coupling, respectively. The extracted mean square displacements also reveal a greater motional flexibility of IDP compared with globular, folded proteins and more restricted water motions on the IDP surface. The results provide evidence that protein and hydration-water motions mutually affect and shape each other, and that there is a gradient of coupling across different protein classes that may play a functional role in macromolecular activity in a cellular context.     

Abnormal solubility behavior of beta-lactoglobulin: salting-in by glycine and NaCl

The causes of the salting-in of beta-lactoglobulin by glycine and NaCl, a solubility behavior contrary to expectations, were probed by a detailed study of the interactions between these solvent components and the protein. The preferential interactions of beta-lactoglobulin with solvent components in aqueous glycine and NaCl systems have been compared with those of bovine serum albumin and lysozyme. At neutral pH, beta-lactoglobulin exhibited insignificant preferential interactions in glycine and NaCl at low cosolvent concentrations and an increasing preferential hydration at higher concentrations, the levels approaching the values expected from the other two proteins. These results indicate considerable binding of the electrolytes to beta-lactoglobulin, sufficient to compensate for the exclusion due to perturbation of the solvent surface tension. The difference between the preferential interactions of beta-lactoglobulin and the other proteins with these two solvent additives was shown to be the cause of the increase of beta-lactoglobulin solubility even at high concentrations of the additives, at which they have salting-out effects on the other proteins. The preferential interactions of NaCl with the three proteins were examined as a function of pH. The results showed no pH dependence of the preferential hydration for bovine serum albumin and lysozyme, while this parameter increased significantly for beta-lactoglobulin at lower pH. This suggests that the binding of electrolytes to beta-lactoglobulin is due to a unique charge distribution on the surface of the protein around neutral pH, which imparts to this protein a large dipole moment.     

Effects of anion substitution on hydration behavior and water uptake of the red-spotted toad, Bufo punctatus: is there an anion paradox in amphibian skin?

Amphibians absorb water osmotically across their skins and rely on chemosensory information from the skin to assess the suitability of hydration sources. The time spent with skin in contact with a moist surface provides a quantitative measure of their ability to perceive the ionic and osmotic properties of aqueous solutions. Dehydrated toads given hyperosmotic (250 mM) solutions of NaCl or Na-gluconate showed significantly longer periods of hydration behavior on the gluconate solution, but they lost water osmotically when immersed in either solution. Similarly, dehydrated toads given 250 mM solutions of NaCl, Na-acetate, Na-phosphate or Na-gluconate showed a progressively greater length of hydration time on solutions with the larger mol. wt anions. These results are consistent with the chemosensory phenomenon previously described in mammalian tongue as 'anion paradox'. On dilute (50 mM) solutions of NaCl or Na-gluconate, the hydration time was not different between anions, despite toads gaining water more rapidly when immersed in dilute NaCl than in Na-gluconate solutions. The differing behavioral results with hyperosmotic and hypoosmotic salt solutions suggest that chemosensory transduction through toad skin involves both transcellular and paracellular pathways.     

Identifying long-range structure in the intrinsically unstructured transactivation domain of p53

Paramagnetic relaxation enhancement (PRE) was used to identify a compact dynamic structure for the intrinsically unstructured transactivation domain of the tumor suppressor protein, p53. Our results show that p53 residues essential for binding to the ubiquitin ligase, MDM2, and the 70 kDa subunit of replication protein A, RPA70, are separated by an average distance of 10-15 A. This result suggests that a more extended member of the ensemble must be populated prior to binding either MDM2 or RPA70. We also show that PRE can be used to detect intermolecular distances between p53 and RPA70.     

Esterase catalysis of substrate vapour: enzyme activity occurs at very low hydration

It has been generally accepted that enzyme activity requires a minimal hydration of about 0.2 g H2O g(-1) protein. This fits well with evidence that hydration above this level is associated with the onset of intramolecular motions. The influence of enzyme hydration on the hydrolysis of substrate by Candida rugosa Lipase B and pig liver esterase was investigated. Each enzyme was studied as a powder at various hydration levels, using vapour phase ethyl butyrate as substrate. This procedure allows the separation of those effects that are due to hydration from those arising from diffusional constraints. We found hydrolytic activity in both enzymes at all hydration levels above zero (between 0.054-0.47 and 0.029-0.60 g H2O g(-1) protein, respectively) that were investigated. The lowest hydration level investigated, <0.03 g H2O g(-1) enzyme, corresponded to a water/enzyme mole ratio of 100 and a coverage of about 10% of the enzyme surface by water molecules. The hydrolytic activity of both enzymes was dependent on protein hydration. However, since the hydrolysis of ethyl butyrate requires water as a second substrate, the absence of activity at zero hydration does not rule out the possibility of enzyme activity in the absence of water. These results suggest that the properties conferred on proteins by water, at least above 10% surface coverage (in this case corresponding to a hydration level of 0.03 g H2O g(-1) protein), are not a requirement for enzyme catalysis.     

Intrinsic dynamics of the partly unstructured PX domain from the Sendai virus RNA polymerase cofactor P

Despite their evident importance for function, dynamics of intrinsically unstructured proteins are poorly understood. Sendai virus phosphoprotein, cofactor of the RNA polymerase, contains a partly unstructured protein domain. The phosphoprotein X domain (PX) is responsible for binding the polymerase to the nucleocapsid assembling the viral RNA. For RNA synthesis, the interplay of the dynamics of the unstructured and structured PX subdomains is thought to drive progression of the RNA polymerase along the nucleocapsid. Here we present a detailed study of the dynamics of PX using hydrogen/deuterium exchange and different NMR relaxation measurements. In the unstructured subdomain, large amplitude fast motions were found to be fine-tuned by the presence of residues with short side chains. In the structured subdomain, where fast motions of both backbone and side chains are fairly restricted, the first helix undergoes slow conformational exchange corresponding to a local unfolding event. The other two helices, which represent the nucleocapsid binding site, were found to be more stable and to reorient with respect to each other, as probed by slow conformational exchange identified for residues on the third helix. The study illustrates the intrinsically differential dynamics of this partly unstructured protein and proposes the relation between these dynamics and its function.     

Conformation-specific binding of alpha-synuclein to novel protein partners detected by phage display and NMR spectroscopy

Alpha-synuclein (AS) is an intrinsically unstructured protein in aqueous solution but is capable of forming beta-sheet-rich fibrils that accumulate as intracytoplasmic inclusions in Parkinson disease and certain other neurological disorders. However, AS binding to phospholipid membranes leads to a distinct change in protein conformation, stabilizing an extended amphipathic alpha-helical domain reminiscent of the exchangeable apolipoproteins. To better understand the significance of this conformational change, we devised a novel bacteriophage display screen to identify protein binding partners of helical AS and have identified 20 proteins with roles in diverse cellular processes related to membrane trafficking, ion channel modulation, redox metabolism, and gene regulation. To verify that the screen identifies proteins with specificity for helical AS, we further characterized one of these candidates, endosulfine alpha (ENSA), a small cAMP-regulated phosphoprotein implicated in the regulation of insulin secretion but also expressed abundantly in the brain. We used solution NMR to probe the interaction between ENSA and AS on the surface of SDS micelles. Chemical shift perturbation mapping experiments indicate that ENSA interacts specifically with residues in the N-terminal helical domain of AS in the presence of SDS but not in aqueous buffer lacking SDS. The ENSA-related protein ARPP-19 (cAMP-regulated phosphoprotein 19) also displays specific interactions with helical AS. These results confirm that the helical N terminus of AS can mediate specific interactions with other proteins and suggest that membrane binding may regulate the physiological activity of AS in vivo.     

A careful disorderliness in the proteome: sites for interaction and targets for future therapies

The community of scientists interested in studying intrinsically unstructured (or disordered) proteins has emerged in recent years. What began as a controversial idea has become an established phenomenon. The new, greater focus on proteins that are in some way normally unstructured promises to provide a greater understanding of protein function, particularly with respect to protein-protein interactions. These regions also offer new possibilities into how interactions can be targeted by small molecules.     

Decomposition of protein experimental compressibility into intrinsic and hydration shell contributions

The experimental determination of protein compressibility reflects both the protein intrinsic compressibility and the difference between the compressibility of water in the protein hydration shell and bulk water. We use molecular dynamics simulations to explore the dependence of the isothermal compressibility of the hydration shell surrounding globular proteins on differential contributions from charged, polar, and apolar protein-water interfaces. The compressibility of water in the protein hydration shell is accounted for by a linear combination of contributions from charged, polar, and apolar solvent-accessible surfaces. The results provide a formula for the deconvolution of experimental data into intrinsic and hydration contributions when a protein of known structure is investigated. The physical basis for the model is the variation in water density shown by the surface-specific radial distribution functions of water molecules around globular proteins. The compressibility of water hydrating charged atoms is lower than bulk water compressibility, the compressibility of water hydrating apolar atoms is somewhat larger than bulk water compressibility, and the compressibility of water around polar atoms is about the same as the compressibility of bulk water. We also assess whether hydration water compressibility determined from small compound data can be used to estimate the compressibility of hydration water surrounding proteins. The results, based on an analysis from four dipeptide solutions, indicate that small compound data cannot be used directly to estimate the compressibility of hydration water surrounding proteins.     

Dynamics of uncrystallized water and protein in hydrated elastin studied by thermal and dielectric techniques

Dynamics of uncrystallized water and protein was studied in hydrated pellets of the fibrous protein elastin in a wide hydration range (0 to 23 wt.%), by differential scanning calorimetry (DSC), thermally stimulated depolarization current technique (TSDC) and dielectric relaxation spectroscopy (DRS). Additionally, water equilibrium sorption–desorption measurements (ESI) were performed at room temperature. The glass transition of the system was studied by DSC and its complex dependence on hydration water was verified. A critical water fraction of about 18 wt.% was found, associated with a reorganization of water in the material. Three dielectric relaxations, associated to dynamics related to distinct uncrystallized water populations, were recorded by TSDC and DRS. The low temperature secondary relaxation of hydrophilic polar groups on the protein surface triggered by hydration water for almost dry samples contains contributions from water molecules themselves at higher water fractions (ν relaxation). This particular relaxation is attributed to water molecules in the primary and secondary hydration shells of the protein fibers. At higher temperatures and for water fraction values equal to or higher than 10 wt.%, a local relaxation of water molecules condensed within small openings in the interior of the protein fibers was recorded. The evolution of this relaxation (w relaxation) with hydration level results in enhanced cooperativity at high water fraction values, implying the existence of “internal” water confined within the protein structure. At higher temperatures a relaxation associated with water dynamics within clusters between fibers (p relaxation) was also recorded, in the same hydration range.