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Wide-line 1 H NMR measurements were extended and all results were interpreted in a thermodynamics-based new approach on aqueous solutions of thymosin-β4 (Tβ4 ), stabilin cytoplasmic domain (CTD), and their 1 : 1 complex. Energy distributions of potential barriers controlling the motion of protein-bound water molecules were determined. Heterogeneous and homogeneous regions were found in the protein-water interface. The measure of heterogeneity of this interface gives quantitative value for the portion of disordered parts in the protein. Ordered structural elements were found extending up to ∼20 % of the individual whole proteins. About 40 % of the binding sites of free Tβ4 get involved in bonds holding the complex together. The complex has the most heterogeneous solvent accessible surface (SAS) in terms of protein-water interactions. The complex is more disordered than Tβ4 or stabilin CTD. The greater SAS area of the complex is interpreted as a clear sign of its open structure.

Wide-line (1) H NMR measurements were extended and all results were reinterpreted in a new thermodynamics-based approach to study aqueous solutions of thymosin-beta4 (Tbeta4 ), stabilin C-terminal domain (CTD) and their 1:1 complex. The energy distributions of the potential barriers, which control motion of protein-bound water molecules, were determined. Heterogeneous and homogeneous regions were found at the protein-water interface. The measure of heterogeneity gives a quantitative value for the portion of disordered parts in the protein. Ordered structural elements were found extending up to 20 % of the whole proteins. About 40 % of the binding sites of free Tbeta4 become involved in bonds holding the complex together. The complex has the most heterogeneous solvent accessible surface (SAS) in terms of protein-water interactions. The complex is more disordered than Tbeta4 or stabilin CTD. The greater SAS area of the complex is interpreted as a clear sign of its open structure.

The potential barriers governing the motions of &alpha, synuclein (&alpha, S) variants&rsquo, hydration water, especially energetics of them, is in the focus of the work. The thermodynamical approach yielded essential information about distributions and heights of the potential barriers. The proteins&rsquo, structural disorder was measured by ratios of heterogeneous water-binding interfaces. They showed the &alpha, S monomers, oligomers and amyloids to possess secondary structural elements, although monomers are intrinsically disordered. Despite their disordered nature, monomers have 33% secondary structure, and therefore they are more compact than a random coil. At the lowest potential barriers with mobile hydration water, monomers are already functional, a monolayer of mobile hydration water is surrounding them. Monomers realize all possible hydrogen bonds with the solvent water. &alpha, S oligomers and amyloids have half of the mobile hydration water amount than monomers because aggregation involves less mobile hydration. The solvent-accessible surface of the oligomers is ordered or homogenous in its interactions with water to 66%. As a contrast, &alpha, S amyloids are disordered or heterogeneous to 75% of their solvent accessible surface and both wild type and A53T amyloids show identical, low-level hydration. Mobile water molecules in the first hydration shell of amyloids are the weakest bound compared to other forms.

Wide-line (1) H NMR measurements were extended and all results were reinterpreted in a new thermodynamics-based approach to study aqueous solutions of thymosin-beta4 (Tbeta4 ), stabilin C-terminal domain (CTD) and their 1:1 complex. The energy distributions of the potential barriers, which control motion of protein-bound water molecules, were determined. Heterogeneous and homogeneous regions were found at the protein-water interface. The measure of heterogeneity gives a quantitative value for the portion of disordered parts in the protein. Ordered structural elements were found extending up to 20 % of the whole proteins. About 40 % of the binding sites of free Tbeta4 become involved in bonds holding the complex together. The complex has the most heterogeneous solvent accessible surface (SAS) in terms of protein-water interactions. The complex is more disordered than Tbeta4 or stabilin CTD. The greater SAS area of the complex is interpreted as a clear sign of its open structure.

Here we present a novel method for the characterization of the hydration of protein solutions based on measuring and evaluating two-component wide-line 1H NMR signals. We also provide a description of key elements of the procedure conceived for the thermodynamic interpretation of such results. These interdependent experimental and theoretical treatments provide direct experimental insight into the potential energy surface of proteins. The utility of our approach is demonstrated through the examples of two proteins of distinct structural classes: the globular, structured ubiquitin, and the intrinsically disordered ERD10 (early response to dehydration 10). We provide a detailed analysis and interpretation of data recorded earlier by cooling and slowly warming the protein solutions through thermal equilibrium states. We introduce and use order parameters that can be thus derived to characterize the distribution of potential energy barriers inhibiting the movement of water molecules bound to the surface of the protein. Our results enable a quantitative description of the ratio of ordered and disordered parts of proteins, and of the energy relations of protein–water bonds in aqueous solutions of the proteins.

The experimental characterization of hindered-rotation barriers and mapping the energetic heterogeneity of water molecules bound to the molecular “surface” of proteins is critical for understanding the functional interaction of proteins with their environment. Here, we show how to achieve this goal by an original wide-line NMR procedure, which is based on the spectral motional narrowing phenomenon following the melting (thawing) process of interfacial ice. The procedure highlights the differences between globular and intrinsically disordered proteins and it enables to delineate the effect of solvent on protein structure, making a distinction between point mutants, monomeric and oligomeric states, and characterizing the molecular interactions taking part in different cellular processes. We put this unique experimental approach introducing novel physical quantities and quantifying the heterogeneous distribution of motional activation energy of water in the interfacial landscape into a historical perspective, demonstrating its utility through a variety of globular and disordered proteins.

Human nucleolar phosphoprotein p140 (hNopp 140) is a highly phosphorylated protein inhibitor of casein kinase 2 (CK2). As in the case of many kinase-inhibitor systems, the inhibitor has been described to belong to the family of intrinsically disordered proteins (IDPs), which often utilize transient structural elements to bind their cognate enzyme. Here we investigated the structural status of this protein both to provide distinct lines of evidence for its disorder and to point out its transient structure potentially involved in interactions and also its tendency to aggregate. Structural disorder of hNopp140 is apparent by its anomalous electrophoretic mobility, protease sensitivity, heat stability, hydrodynamic behavior on size-exclusion chromatography, (1)H NMR spectrum and differential scanning calorimetry scan. hNopp140 has a significant tendency to aggregate and the change of its circular dichroism spectrum in the presence of 0-80% TFE suggests a tendency to form local helical structures. Wide-line NMR measurements suggest the overall disordered character of the protein. In all, our data suggest that this protein falls into the pre-molten globule state of IDPs, with a significant tendency to become ordered in the presence of its partner as demonstrated in the presence of transcription factor IIB (TFIIB).

Wide-line (1)H NMR signal intensity, spin-lattice and spin-spin relaxation rates and differential scanning calorimetry (DSC) measurements were done on avian (chicken and turkey) crystalline lenses between -70 degrees C and +45 degrees C to provide quantitative measures of protein hydration characteristic of the protein-water interfacial region. These measures are of paramount importance in understanding both the physiology of crystalline lens and its transitions to the cataractous pathological state characterized by the formation of opaque protein aggregates. Water mobility shows a characteristic transition at about -60 degrees C, which is identified as the melting of the interfacial/hydrate water. The amount of water in the low-temperature mobile fraction is about h = 0.4 g water/g protein, which equals the hydration required for protein activity. The amount of mobile water is temperature-independent up to about -10 degrees C, with a significant increase at higher temperatures below 0 degrees C. Above 0 degrees C, the relaxation processes can be described by a single (for spin-lattice) and by a triple (for spin-spin relaxation) exponential function. The spin-spin relaxation rate component of R(2) = 10-20 s(-1) and its dynamical parameters characterize the interfacial water at ambient or physiological temperatures. When considered an independent phase, the specific heat of the hydrate water obtained by a combination of DSC and NMR data in the temperature range -43 degrees C to -28 degrees C is higher than that of pure/bulk water. This discrepancy can only be resolved by assuming that the hydrate water is in strong thermodynamic coupling with the protein matrix. The specific heat for the system composed of the protein molecule and its hydration water is 4.6 +/- 0.3 J g(-1) K(-1). Thus, in a thermodynamic sense, crystalline protein and its hydrate layer behave as a highly-interconnected single phase.

Protein disorder is predicted to be widespread in eukaryotic proteomes, although direct experimental evidence is rather limited so far. To fill this gap and to unveil the identity of novel intrinsically disordered proteins (IDPs), proteomic methods that combine 2D electrophoresis with mass spectrometry have been developed. Here, we applied the method developed in our laboratory [ Csizmok et al., Mol. Cell. Proteomics 2006, 5, 265- 273 ] to the proteome of Drosophila melanogaster. Protein Df31, earlier described as a histone chaperone involved in chromatin decondensation and stabilization, was among the IDPs identified. Despite some hints at the unusual structural behavior of Df31, this protein has not yet been structurally characterized. Here, we provide evidence by a variety of techniques such as CD, NMR, gel-filtration, limited proteolyzsis and bioinformatics that Df31 is intrinsically disordered along its entire length. Further, by chemical cross-linking, we provide evidence that it is a monomeric protein, and suggest that its function(s) may benefit from having an extended and highly flexible structural state. The potential functional advantages and the generality of protein disorder among chromatin organizing proteins are discussed in detail. Finally, we also would like to point out the utility of our 2DE/MS technique for discoveringor, as a matter of fact, rediscoveringIDPs even from the complicated proteome of an advanced eukaryote.

The fraction of mobile (unfrozen) water during phase transitions of NaCl–H2O solution and tris(hydroxymethyl)aminomethane (Tris) buffer solution has been determined by differential scanning calorimetry (DSC). Measurements were carried out in the temperature range between −50 and +30 ◦ C with a heating rate of 2 K min −1 . The fraction of mobile water was estimated from the enthalpy of melting of the different frozen phases present in aqueous solution samples. Results are supplemented by proton NMR intensity measurements in the same temperature range. A small endothermic peak was detected in the DSC curves at the same temperature where the change in the NMR intensity occurs. We assume that activation/deactivation of rotational molecular motion of hydration water molecules occurs at the steps of the NMR intensity at higher and lower temperatures during heating and cooling, respectively. The rotational motion is probably the initial stage of the eutectic phase separation. Tris additive to NaCl solution causes thermal shifts of the small endothermic peak and in the related NMR intensity. These qualitative differences indicate interactions of Na + and Cl − ions with the small organic molecule constituents.

In this work, the electrochemical hydrogen doping and withdrawal process of Pd metal was examined with respect to the applicability of Faraday’s law. It was found that after a suitable electrolytic pre-treatment, the normalized resistivity ( R / R 0 ) versus hydrogen concentration ( c F = H/M) curves where c F was determined from Faraday’s law by assuming that the 100% current efficiency is independent of the value of the applied charging current density in a wide range. In the α-Pd(H) phase (up to c (α max ) = 0.015H/M), a good quantitative agreement was found with previous resistivity studies by both electrolytic and gas-phase charging. However, for c > c (α max ) our R / R 0 versus c F data did not agree with the R / R 0 versus c H curve from previously reported electrochemical charging experiments of Pd metal where the doped amount of hydrogen, c H , was determined by direct methods. The two sets of resistivity data can be brought into good agreement by assuming that the current efficiency for the absorption process is η a = 1.55. Similarly, for the electrolytic desorption process, an agreement with the previously reported R / R 0 versus c H curve could be achieved by taking η d = 1.9. These current efficiency values higher than unity would imply that each hydrogen absorbed/desorbed by electrolytic charge/discharge processes is accompanied by the absorption/desorption of some excess H atoms by processes not requiring electric charge transfer (i.e., only neutral H atoms are involved). At present, we do not have a physical/chemical picture about the nature of such processes.

The partition of hydrogen into diffusible and residual parts was realized by pulse NMR spectroscopy, by gas chromatography and by prompt gamma activation analysis (PGAA). The total hydrogen content was determined by the two non-NMR methods and the diffusible (mobile) component by CPMG NMR pulse sequence. Results on amorphous Ni(Cu)–Zr–H systems of different compositions are shown. Partially crystallized samples were also studied as an extension. A method proposed by us directly gives the fractional population of hydrogen atoms in the free (mobile) state on the spin–spin relaxation time scale. On the other hand the least values of the residual hydrogen content correlate surprisingly well with the numbers of filled four Zr-type H-sites calculated by Batalla et al. [NATO ASI Ser. 136 (1985) 203] for 0.21-nm exclusion distance.

We have investigated the rotor-stator molecular crystal fullerene-cubane (C 60 .C 8 H 8 ) by 1 H'NMR spectroscopy in the temperature range of 130 to 380 K. The 1 H line is broad and exhibits fine structure characteristic of a multispin spectrum, indicating the absence of angular reorientations of the cubane molecule. The temperature and field dependence of the spin-lattice relaxation rate of cubane protons is well explained in terms of field fluctuations arising from reorientations of the C 60 molecules. Despite the closely packed structure, the activation energy of these reorientations, 650 K, is close to that observed in the high-temperature phase of pure C 60 indicating that the static cubane molecules act as efficient bearing for the rotating fullerene molecules.

Our work proves that positron annihilation spectroscopy is an excellent tool to follow the structural changes in chemical species. We present four examples to support the above statement. The sizes of defects in electrodeposited chromium layers were studied and estimated on the basis of positron lifetime spectra decomposed into two components. Vacancies, di-vacancies and vacancy-clusters could be identified in the electrodeposites. The changes of the size distribution of the free volume units in poly(methylmetacrylate) on the dependence of molecular weight and dispersity were described by the correlation between the lifetime ofortho-Ps and the free volume units in polymers. It was found that the free volume is affected by both the molecular weight and dispersity. The effect of dispersity was explained by the sample preparation technique, namely by the application of high pressure. The ortho-para conversion ofortho-Ps was used to follow the partial spin-crossover in [Fe(1-ethyl-1H-tetrazole)6](BF4)2. The spin-crossover temperature was identified to be 105 K. A conformal structural transformation was found in [Zn(1-propyl-1H-tetrazole)6](BF4)2 between 170 and 90 K by positronium lifetime measurement and the role of (BF4)2− anion, in this transformation, was proved by19F NMR spectroscopy.

The splat-quenched Ni50Zr50 alloy has a rosette-shaped phase with tenfold symmetry. The structure of this phase has been interpreted (Yang, 1985) as “tenfold twins” of the orthorhombic NiZr phase. The formation and the feature of the phase with tenfold symmetry have been investigated in Ni50Zr50, Ni50−xCuxZr50 and Ni50−xCoxZr50 amorphous alloys. It was found that this phase forms first during the crystallization process. The structure comes nearer to the structure of a quasi-crystal through the influence of Cu and Co atoms.

CASK-interactive protein1 is a newly recognized post-synaptic density protein in mammalian neurons. Although its N-terminal region contains several well-known functional domains, its entire C-terminal proline-rich region of 800 amino acids lacks detectable sequence homology to any previously characterized protein. We used multiple techniques for the structural characterization of this region and its three fragments. By bioinformatics predictions, CD spectroscopy, wide-line and 1H-NMR spectroscopy, limited proteolysis and gel filtration chromatography, we provided evidence that the entire proline-rich region of CASK-interactive protein1 is intrinsically disordered. We also showed that the proline-rich region is biochemically functional, as it interacts with the adaptor protein Abl-interactor-2. To extend the finding of a high level of disorder in this scaffold protein, we collected 74 scaffold proteins (also including proteins denoted as anchor and docking), and predicted their disorder by three different algorithms. We found that a very high fraction (53.6% on average) of the residues fall into local disorder and their ordered domains are connected by linker regions which are mostly disordered (64.5% on average). Because of this high frequency of disorder, the usual design of scaffold proteins of short globular domains (86 amino acids on average) connected by longer linker regions (140 amino acids on average) and the noted binding functions of these regions in both CASK-interactive protein1 and the other proteins studied, we suggest that structurally disordered regions prevail and play key recognition roles in scaffold proteins. Structured digital abstract • MINT-7034649: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with L1CAM (uniprotkb:P32004) by two hybrid (MI:0018) • MINT-7034677: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with NCK1 (uniprotkb:P16333) by two hybrid (MI:0018) • MINT-7034706: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with Stathmin-3 (uniprotkb:Q9NZ72) by two hybrid (MI:0018) • MINT-7034579: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with ABI2 (uniprotkb:Q9NYB9) by two hybrid (MI:0018) • MINT-7034720: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with Synaptotagmin (uniprotkb:P21579) by two hybrid (MI:0018) • MINT-7034691: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with Neurexin-2 (uniprotkb:Q9P2S2) by two hybrid (MI:0018) • MINT-7034617: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with CASK (uniprotkb:P07498) by two hybrid (MI:0018) • MINT-7034748: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with SIAH1 (uniprotkb:Q8IUQ4) by two hybrid (MI:0018) • MINT-7034663: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with Myosin-Ib (uniprotkb:O43795) by two hybrid (MI:0018) • MINT-7034734: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with Septin-4 (uniprotkb:O43236) by two hybrid (MI:0018) • MINT-7034634: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with EPHA2 (uniprotkb:P29317) by two hybrid (MI:0018) • MINT-7034765, MINT-7034783: Caskin1 (uniprotkb:Q8VHK2) physically interacts (MI:0915) with ABI2 (uniprotkb:Q9NYB9) by pull down (MI:0096)

In vitro 1H pulse nuclear magnetic resonance (NMR) studies on human intervertebral disc tissues were performed to establish the characteristics of relaxation processes. The spin-lattice relaxation time, bound water and dry matter content were measured. Biopsy material from fresh cadavers was taken from different discs along the spinal column and from different sectors of some single discs. It is suggested that the measured parameters correlate with the biomechanical behavior of discs, which in turn is influenced by function, by exposure to physical stresses, or by aging.