K. Tompa

The state of water in normal and senile cataractous lenses studied by nuclear magnetic resonance

Normal and senile cataractous lenses were studied by nuclear magnetic resonance in order to get information about the state of water during the progression of cataract. Water proton spin-spin (T2) and spin-lattice relaxation times—in the laboratory and the rotating frame—() were measured at 34 and 90 MHz. At temperatures below −9°C the relaxation times of protons in “non-freezable” component of lens water were measured as a function of the temperature, and T1 and T2 relaxation times were found to be the same for both the normal and the cataractous lenses. At temperatures above −9°C a marked difference in relaxation times T1 and T2 was found between normal and completely opaque lenses. It was concluded that the most characteristic difference between the normal and completely opaque lenses should be accounted for by the change in the “bound”-to-“free” ratio of lens 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. 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.

The state of water in normal human, bird and fish eye lenses

Abstract The state of water was studied in normal human, fish and bird eye lenses. Free induction decay as well as nuclear spin-lattice and nuclear spin-spin relaxation time measurements were carried out by NMR method. The amount of non-freezable water per unit protein quantity was determined and the activation energy of the motion of the water molecules responsible for the relaxation was estimated. These results were compared with the ideas of Kuch and Yu concerning difference in protein conformation of the lens crystallins.

Water fractions in normal and senile cataractous eye lenses studied by NMR

The state of water and water fractions in normal and senile cataractous eye lenses was studied by the NMR method. Combining NMR with vacuum dehydration provided additional information on multifractional samples. A new mathematical procedure is presented which separates the characteristic parameters of the different fractions and helps to determine the relaxation times and amounts of the fractions. The measurement accuracy enables separation of three different water fractions both in normal and in cataractous lenses.

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. The amount of water in the low-temperature mobile fraction is 0.4 g/g protein for the pure water solution. The amount of mobile water is higher and its temperature dependence more pronounced for the buffered solution. The specific heat of the nonfreezing/hydrate water was evaluated using combined differential scanning calorimetry and NMR data. Considering the interfacial region as an independent phase, the values obtained are 5.0-5.8 J x g(-1) x K(-1), and the magnitudes are higher than that of pure/bulk water (4.2 J x g(-1) x K(-1)). This unexpected discrepancy can only be resolved in principle by assuming that hydrate water is in tight H-bond coupling with the protein matrix. The specific heat for the system composed of the protein molecule and its hydration water is 2.3 J x g(-1) x K(-1). It could be concluded that the protein ubiquitin and its hydrate layer behave as a highly interconnected single phase in a thermodynamic sense.

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 Parkinsons disease. Three familial mutations, A30P, E46K, and A53T, correlate with Parkinsons disease. Therefore, unraveling the structural effects of these mutations has basic implications in understanding the molecular basis of the disease. Here, we address this issue through comparing details of the hydration of wild-type α-synuclein and its A53T mutant by a combination of wide-line NMR, differential scanning calorimetry, and molecular dynamics simulations. All three approaches suggest a hydrate shell compatible with a largely disordered state of both proteins. Its fine details, however, are different, with the mutant displaying a somewhat higher level of hydration, suggesting a bias to more open structures, favorable for protein-protein interactions leading to amyloid formation. These differences disappear in the amyloid state, suggesting basically the same surface topology, irrespective of the initial monomeric state.

Diffusible and residual hydrogen in amorphous Ni(Cu)-Zr-H alloys

Abstract 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.

C.P.-D.D.-M.A.S. 13C-N.M.R. investigations of anhydrous and hydrated cyclomalto-oligosaccharides: The role of water of hydration

Abstract Hydrated and anhydrous cyclomaltohexaose, cyclomaltoheptaose, and cyclomalto-octaose (cyclodextrins) have been investigated by the c.p.-d.d.-m.a.s. 13 C-n.m.r. technique. The chemical shifts of the signals of C-1 and C-6 provide information about conformation and the results agree fairly well with the earlier scattering data on hydrated systems, but some discrepancies have been found for cyclomaltohexaose. Th conformation of the macro-rings seems to be determined by the hydration water. The unique role of water in forming crystals of cyclomalto-oligosaccharides is demonstrated.

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., 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.

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. 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).