Pawel Kamasa

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.

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.

Analysis of Alloys Using DTA and TD Methods with Simultaneous Thermomagnetic Studies

A modified thermoanalyzer for investigation ferromagnetic materials as metals and alloys is described. Conventional analog phase detection technique is replaced by specialized digital one which added some new outstanding features of the instrumentation. Samples of the material can be investigated simultaneously by three methods: differential thermal analysis (DTA), thermal dilatometry (TD) and thermomagnetometry (TMAG).

Application of dilatometry with modulated temperature for thermomechanical analysis of anti-wear coating/substrate systems

Abstract Modern vacuum-plasma surface modification technologies of tools for metal and wood machining are based inter alia on the deposition of thin gradient and multilayer protection coatings with a thickness of several micrometers. Exploitation of tools are frequently carried out in complex tribological nodes, where next to mechanical loads, there are significant changes in temperature at the interface of the tool/workpiece material; hence, the knowledge of the thermomechanical stability of the coatings operating under these conditions is an important criterion used in the processes of designing of wear-resistant coating technology. In this paper, the objects of research were three types of gradient PVD coating/molybdenum substrate systems, for which time courses of amplitudes of elongation and equivalent thermal expansion coefficients of the systems were measured. Experimental measurements were made using a temperature-modulated dilatometry using concepts of dynamic load thermomechanical analysis. It was demonstrated that the developed method allows a quantitative determination of the influence of the gradient layer type on the total elongation of the substrate of the system. It was also shown that the changes of thermomechanical properties of the systems during annealing process are correlated with the time evolution of the equivalent thermal expansion coefficient. In addition, using the concepts of transition function describing the continuous change of physical and chemical parameters as a function of the spatial variables and finite element method for each of the systems, the distributions of internal thermal stresses were determined.

Irreversing thermal expansivity of materials coated with adhesive thin films detected by modulated-temperature dilatometry and differential thermal analysis

Usability of simultaneous modulated-temperature dilatometry (MT DIL) and modulated-temperature differential thermal analysis (MT DTA) for the quantitative description of degradation kinetics of wear-resistant film adhesion to the substrate as a function of temperature are presented in this work. The reversing thermal expansion coefficient of materials as a parameter sensitive to the relaxation of mechanical thermal stresses in films tested was used for this purpose. Sample experimental results allowing the process of film degradation to be described as a function of increasing temperature are quoted. Usefulness of this method considering the films deposited at diversified process parameters is also substantiated.

Application of Temperature Modulated Relative Dilatometry. Temperatures of adhesion degradation

Application of temperature modulated dilatometry (TM DIL) to investigation on degradation of the adhesion between ceramic films and the substrate is presented. Layers of titanium nitride deposited by plasma assisted physical vapour deposition (PA PVD) methods on the Armco iron substrates were tested. This paper shows that the TM DIL method is helpful in determining the usefulness of the titanium nitride covering of the cutting tools and machine parts.

Temperature modulation in dilatometric investigation of PVD substrate-coating systems

Temperature modulation technique and phase sensitive analysis is applied for investigation the thermal stresses induced at higher temperature in the space between coating and substrate. The method applying only phase shift parameter eliminates influence of the intensity variation of measured signal. Comparison with conventional application of temperature modulation is presented. Method gives comprehensive information about the state of coating which is usually immersed using amplitude analysis.

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.