Mónika Zsuzsanna Bokor

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.

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.

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).

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. 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.

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.1 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. Although the cellular readout of these interactions and the formation of these complexes have been thoroughly described, no attempt was made to study these interactions in detail, and to elucidate the thermodynamic, kinetic, and structural underpinning of this range of moonlighting functions. Because Tβ4 is mostly disordered, and its 4 described partners are structurally unrelated (the CTD of stabilin-2 is actually fully disordered), it occurred to us that this system might be ideal to characterize the structural adaptability and ensuing moonlighting functions of IDPs. Unexpectedly, we found that Tβ4 engages in multiple weak, transient, and fuzzy interactions, i.e., it is capable of mediating distinct yet specific interactions without adapting stable folded structures.

Hydrogen mobility and protein-water interaction in proteins of 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 1 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 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).