WT and A53T α-Synuclein Systems: Melting Diagram and Its New Interpretation.

The potential barriers governing the motions of -synuclein (S) variants' 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' structural disorder was measured by ratios of heterogeneous water-binding interfaces.

Molecular Motions and Interactions in Aqueous Solutions of Thymosin-β , Stabilin CTD and Their 1 : 1 Complex, Studied by H-NMR Spectroscopy.

Wide-line H NMR measurements were extended and all results were interpreted in a thermodynamics-based new approach on aqueous solutions of thymosin-β (Tβ ), 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.

Hydration shell differentiates folded and disordered states of a Trp-cage miniprotein, allowing characterization of structural heterogeneity by wide-line NMR measurements.

Hydration properties of folded and unfolded/disordered miniproteins were monitored in frozen solutions by wide-line H-NMR. The amount of mobile water as function of T (-80 °C < T < 0 °C) was found characteristically different for folded (TC5b), semi-folded (pH < 3, TCb5(H+)) and disordered (TC5b_N1R) variants. Comparing results of wide-line H-NMR and molecular dynamics simulations we found that both the amount of mobile water surrounding proteins in ice, as well as their thaw profiles differs significantly as function of the compactness and conformational heterogeneity of their structure.

The Melting Diagram of Protein Solutions and Its Thermodynamic Interpretation.

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.

Molecular Motions and Interactions in Aqueous Solutions of Thymosin-β , Stabilin C-Terminal Domain (CTD) and Their 1:1 Complex Studied by H NMR Spectroscopy.

Wide-line H NMR measurements were extended and all results were reinterpreted in a new thermodynamics-based approach to study aqueous solutions of thymosin-β (Tβ ), 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.

Hydrogen Mobility and Protein-Water Interactions in Proteins in the Solid State.

In this work the groundwork is laid for characterizing the mobility of hydrogen-hydrogen pairs (proton-proton radial vectors) in proteins in the solid state that contain only residual water. In this novel approach, we introduce new ways of analyzing and interpreting data: 1) by representing hydrogen mobility (HM) and melting diagram (MD) data recorded by wide-line H NMR spectroscopic analysis as a function of fundamental temperature (thermal excitation energy); 2) by suggesting a novel mode of interpretation of these parameters that sheds light on details of protein-water interactions, such as the exact amount of water molecules and the distribution of barrier potentials pertaining to their rotational and surface translational mobility; 3) by relying on directly determined physical observables. We illustrate the power of this approach by studying the behavior of two proteins, the structured enzyme lysozyme and the intrinsically disordered ERD14.

Wide-line NMR and DSC studies on intrinsically disordered p53 transactivation domain and its helically pre-structured segment.

Wide-line 1H NMR intensity and differential scanning calorimetry measurements were carried out on the intrinsically disordered 73-residue full transactivation domain (TAD) of the p53 tumor suppressor protein and two peptides: one a wild type p53 TAD peptide with a helix pre-structuring property, and a mutant peptide with a disabled helix-forming propensity. Measurements were carried out in order to characterize their water and ion binding characteristics. By quantifying the number of hydrate water molecules, we provide a microscopic description for the interactions of water with a wild-type p53 TAD and two p53 TAD peptides.

Multiple fuzzy interactions in the moonlighting function of thymosin-β4.

Thymosine β4 (Tß4) is a 43 amino acid long intrinsically disordered protein (IDP), which was initially identified as an actin-binding and sequestering molecule. Later it was described to have multiple other functions, such as regulation of endothelial cell differentiation, blood vessel formation, wound repair, cardiac cell migration, and survival. The various functions of Tβ4 are mediated by interactions with distinct and structurally unrelated partners, such as PINCH, ILK, and stabilin-2, besides the originally identified G-actin.

Hydrogen skeleton, mobility and protein architecture.

The mobility of the proton-proton radial vectors is introduced as a quantitative measure for the structural dynamics of organic materials, especially protein molecules. As defined for the entire molecule, the hydrogen mobility () is proposed as an "order parameter," which describes the effect of motional narrowing on inter-proton dipole-dipole interactions. satisfies all requirements of an order parameter in the Landau molecular field theory of phase transitions.

Structural disorder and local order of hNopp140.

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.

Wide-line NMR and protein hydration.

In this chapter, the reader is introduced to the basics of wide-line NMR, with particular focus on the following: (1) basic theoretical and experimental NMR elements, necessary before switching the spectrometer and designing the experiment, (2) models/theories for the interpretation of measured data, (3) definition of wide-line NMR spectrometry, the description of the measurement and evaluation variants, useful hints for the novice, (4) advice on selecting the solvent, which is not a trivial task, (5) a note of warning that not all data are acceptable in spite of the statistical confidence. Finally, we wrap up the chapter with the results on two proteins (a globular and an intrinsically disordered).

Distinct hydration properties of wild-type and familial point mutant A53T of α-synuclein associated with Parkinson's disease.

The propensity of α-synuclein to form amyloid plays an important role in Parkinson's disease. Three familial mutations, A30P, E46K, and A53T, correlate with Parkinson's disease. Therefore, unraveling the structural effects of these mutations has basic implications in understanding the molecular basis of the disease.

Hydration water/interfacial water in crystalline lens.

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.

High levels of structural disorder in scaffold proteins as exemplified by a novel neuronal protein, CASK-interactive protein1.

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.

Interfacial water at protein surfaces: wide-line NMR and DSC characterization of hydration in ubiquitin solutions.

Wide-line 1H-NMR and differential scanning calorimetry measurements were done in aqueous solutions and on lyophilized samples of human ubiquitin between -70 degrees C and +45 degrees C. The measured properties (size, thermal evolution, and wide-line NMR spectra) of the protein-water interfacial region are substantially different in the double-distilled and buffered-water solutions of ubiquitin. The characteristic transition in water mobility is identified as the melting of the nonfreezing/hydrate water.

Intrinsic structural disorder of DF31, a Drosophila protein of chromatin decondensation and remodeling activities.

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.

Protein-water and protein-buffer interactions in the aqueous solution of an intrinsically unstructured plant dehydrin: NMR intensity and DSC aspects.

Proton NMR intensity and differential scanning calorimetry measurements were carried out on an intrinsically unstructured late embryogenesis abundant protein, ERD10, the globular BSA, and various buffer solutions to characterize water and ion binding of proteins by this novel combination of experimental approaches. By quantifying the number of hydration water molecules, the results demonstrate the interaction between the protein and NaCl and between buffer and NaCl on a microscopic level. The findings overall provide direct evidence that the intrinsically unstructured ERD10 not only has a high hydration capacity but can also bind a large amount of charged solute ions.

Ion-selective supported liquid membranes placed under steady-state diffusion control.

Supported liquid membranes are used here to establish steady-state concentration profiles across ion-selective membranes rapidly and reproducibly. This opens up new avenues in the area of nonequilibrium potentiometry, where reproducible accumulation and depletion processes at ion-selective membranes may be used to gain valuable analytical information about the sample. Until today, drifting signals originating from a slowly developing concentration profile across the ion-selective membrane made such approaches impractical in zero current potentiometry.

Rotor-stator molecular crystals of fullerenes with cubane.

Cubane (C8H8) and fullerene (C60) are famous cage molecules with shapes of platonic or archimedean solids. Their remarkable chemical and solid-state properties have induced great scientific interest. Both materials form polymorphic crystals of molecules with variable orientational ordering.

Primary contact sites in intrinsically unstructured proteins: the case of calpastatin and microtubule-associated protein 2.

Intrinsically unstructured proteins (IUPs) exist in a disordered conformational state, often considered to be equivalent with the random-coil structure. We challenge this simplifying view by limited proteolysis, circular dichroism (CD) spectroscopy, and solid-state (1)H NMR, to show short- and long-range structural organization in two IUPs, the first inhibitory domain of calpastatin (CSD1) and microtubule-associated protein 2c (MAP2c). Proteases of either narrow (trypsin, chymotrypsin, and plasmin) or broad (subtilisin and proteinase K) substrate specificity, applied at very low concentrations, preferentially cleaved both proteins in regions, i.

NMR relaxation studies on the hydrate layer of intrinsically unstructured proteins.

Intrinsically unstructured/disordered proteins (IUPs) exist in a disordered and largely solvent-exposed, still functional, structural state under physiological conditions. As their function is often directly linked with structural disorder, understanding their structure-function relationship in detail is a great challenge to structural biology. In particular, their hydration and residual structure, both closely linked with their mechanism of action, require close attention.

1H spin-spin relaxation in normal and cataractous human, normal fish and bird eye lenses.

A systematic study on nuclear spin-spin relaxation of water protons in human, fish and bird eye lenses/lens nuclei is reported. The purpose of this study is to clarify the real nature of the relaxation processes not describable as a single exponential decay. The characterization of the spin-spin relaxation by a single exponential is commonly used both in literature and in MRI diagnostics.

Variability of magnetic resonance parameters in pituitary adenomas at low temperature.

Temperature dependent spin-lattice relaxation time, T1, measurements were carried out on surgically removed pituitary adenoma tissues at low temperature. Differences were found between samples; the temperature dependence of T1 showed a broad minimum below freezing. The measured data were fitted by theoretical lines to determine various parameters such as activation energy, correlation time and intensity.