"Water Association" Band in Saccharide Amorphous Matrices: Role of Residual Water on Bioprotection.

Saccharides protect biostructures against adverse environmental conditions mainly by preventing large scale motions leading to unfolding. The efficiency of this molecular mechanism, which is higher in trehalose with respect to other sugars, strongly depends on hydration and sugar/protein ratio. Here we report an Infrared Spectroscopy study on dry amorphous matrices of the disaccharides trehalose, maltose, sucrose and lactose, and the trisaccharide raffinose. Samples with and without embedded protein (Myoglobin) are investigated at different sugar/protein ratios, and compared. To inspect matrix properties we analyse the Water Association Band (WAB), and carefully decompose it into sub-bands, since their relative population has been shown to effectively probe water structure and dynamics in different matrices. In this work the analysis is extended to investigate the structure of protein-sugar-water samples, for the first time. Results show that several classes of water molecules can be identified in the protein and sugar environment and that their relative population is dependent on the type of sugar and, most important, on the sugar/protein ratio. This gives relevant information on how the molecular interplay between residual waters, sugar and protein molecules affect the biopreserving properties of saccharides matrices.

Closed-Locked and Apo-Resting State Structures of the Human α7 Nicotinic Receptor: A Computational Study.

Nicotinic acetylcholine receptors, belonging to the Cys-loop superfamily of ligand-gated ion channels (LGICs), are membrane proteins present in neurons and at neuromuscular junctions. They are responsible for signal transmission, and their function is regulated by neurotransmitters, agonists, and antagonists drugs. A detailed knowledge of their conformational transition in response to ligand binding is critical to understanding the basis of ligand-receptor interaction, in view of new pharmacological approaches to control receptor activity. However, the scarcity of experimentally derived structures of human channels makes this perspective extremely challenging. To contribute overcoming this issue, we have recently reported structural models for the open and the desensitized states of the human α7 nicotinic receptor. Here, we provide all-atom structural models of the same receptor in two different nonconductive states. The first structure, built via homology modeling and relaxed with extensive Molecular Dynamics simulations, represents the receptor bound to the natural antagonist α-conotoxin ImI. After comparison with available experimental data and computational models of other eukaryotic LGICs, we deem it consistent with the "closed-locked" state. The second model, obtained with simulations from the spontaneous relaxation of the open, agonist-bound α7 structure after ligand removal, recapitulates the characteristics of the apo-resting state of the receptor. These results add to our previous work on the active and desensitized state conformations, contributing to the structural characterization of the conformational landscape of the human α7 receptor and suggesting benchmarks to discriminate among conformations found in experiments or in simulations of LGICs. In particular key interactions at the interface between the extracellular domain and the transmembrane domain are identified, that could be critical to the α7 receptor function.

Bioprotection Can Be Tuned with a Proper Protein/Saccharide Ratio: The Case of Solid Amorphous Matrices.

Saccharides, and in particular trehalose, are well known for their high efficiency in protecting biostructures against adverse environmental conditions. The protein dynamics is known to be highly inhibited in a low-water trehalose host medium, the inhibition being markedly dependent on the amount of residual water. Besides hydration, the protein/sugar ratio is expected to affect the properties of saccharide amorphous matrices. In this work, we report an infrared spectroscopy study in dry amorphous matrices of various sugars (the disaccharides trehalose, maltose, sucrose, and lactose, and the trisaccharide raffinose) containing myoglobin, at different protein/sugar ratios. We analyze the stretching band of the bound CO molecule and the water association band. Such bands have already been successfully exploited for the simultaneous study of thermal evolution of a matrix and embedded protein. The results show a high dependence of protein and matrix signals on the protein/sugar ratio, the system behavior evolving from situations where (i) the protein slaves the matrix to (ii) protein ↔ matrix coupling/uncoupling, then to (iii) the matrix slaving the protein, with increasing sugar concentration. This supports a mutual protein ↔ matrix structural and dynamic influence in low hydrated systems, indicating that the protein/solvent master and slave paradigm does not strictly hold, but the mutual relationship depends on the relative concentrations. Furthermore, for each sugar, an optimal protein/sugar concentration ratio can be identified, which maximizes the protein preservation; under such a condition, the water content is minimal.

Biopreservation of Myoglobin in Crowded Environment: A Comparison between Gelatin and Trehalose Matrixes.

Biopreservation by sugar and/or polymeric matrixes is a thoroughly studied research topic with wide technological relevance. Ternary amorphous systems containing both saccharides and proteins are extensively exploited to model the in vivo biopreservation process. With the aim of disentangling the effect of saccharides and polypeptidic crowders (such as gelatin) on the preservation of a model protein, we present here a combined differential scanning calorimetry and UV-vis spectrophotometry study on samples of myoglobin embedded in amorphous gelatin and trehalose + gelatin matrixes at different hydrations, and compare them with amorphous myoglobin-only and myoglobin-trehalose samples. The results point out the different effects of gelatin, which acts mainly as a crowding agent, and trehalose, which acts mainly by direct interaction. Gelatin is able to improve effectively the protein thermal stability at very low hydration; however, it has small effects at medium to high hydration. Consistently, gelatin appears to be more effective than trehalose against massive denaturation in the long time range, while the mixed trehalose + collagen matrix is most effective in preserving protein functionality, outdoing both gelatin-only and trehalose-only matrixes.

The water association band as a marker of hydrogen bonds in trehalose amorphous matrices.

The relevant role played by residual water in modulating the dynamics and structure of a protein, a matrix and their coupling has been thoroughly studied in bioprotective amorphous saccharide matrices via experiments and simulations. In order to better characterize this residual water and the hydrogen bond structures in which it is involved, in this work infrared spectroscopy experiments are conducted on trehalose-water systems. The properties of water are inferred from the study of a peculiar infrared band, the water association band, which we exploited as a marker of the hydrogen bonds in which water is involved. Our aim was the identification of populations of water molecules, which give rise to the different components to which the water association band can be easily decomposed. The attribution of these components to families of water molecules is accomplished by studying the band behaviour with a suitable use of Hofmeister salts, known to have a structure-making or structure-breaking activity, and therefore able to modify the hydrogen bond network by enhancing or depressing the local order. The results allow ascribing, in almost all samples, five band components to either a chaotropic or kosmotropic environment, and further define two of them as bulk-like or ice-like water. The characterization of different components enables the use of this band as a tool to deepen the knowledge of other low-water hydrated matrices with a new approach. A differential analysis of peak frequencies and populations of the components in a bulky system, containing or not embedded components or interfaces (e.g. proteins, polymers, surfaces or even massive cosolutes), makes it possible to draw information on the properties of hydrogen bonds which are formed in the investigated systems.

Protein thermal denaturation and matrix glass transition in different protein-trehalose-water systems.

Biopreservation by saccharides is a widely studied issue due to its scientific and technological importance; in particular, ternary amorphous protein-saccharide-water systems are extensively exploited to model the characteristics of the in vivo biopreservation process. We present here a differential scanning calorimetry (DSC) study on amorphous trehalose-water systems with embedded different proteins (myoglobin, lysozyme, BSA, hemoglobin), which differ for charge, surface, and volume properties. In our study, the protein/trehalose molar ratio is kept constant at 1/40, while the water/sugar molar ratio is varied between 2 and 300; results are compared with those obtained for binary trehalose-water systems. DSC upscans offer the possibility of investigating, in the same measurement, the thermodynamic properties of the matrix (glass transition, T(g)) and the functional properties of the encapsulated protein (thermal denaturation, T(den)). At high-to-intermediate hydration, the presence of the proteins increases the glass transition temperature of the encapsulating matrix. The effect mainly depends on size properties, and it can be ascribed to confinement exerted by the protein on the trehalose-water solvent. Conversely, at low hydration, lower T(g) values are measured in the presence of proteins: the lack of water promotes sugar-protein interactions, thus weakening the confinement effect and softening the matrix with respect to the binary system. A parallel T(den) increase is also observed; remarkably, this stabilization can reach ∼70 K at low hydration, a finding potentially of high biotechnological relevance. A linear relationship between T(g) and T(den) is also observed, in line with previous results; this finding suggests that collective water-trehalose interactions, responsible for the glass transition, also influence the protein denaturation.

Myoglobin embedded in saccharide amorphous matrices: water-dependent domains evidenced by small angle X-ray scattering.

We report Small Angle X-ray Scattering (SAXS) measurements performed on samples of carboxy-myoglobin (MbCO) embedded in low-water trehalose glasses. Results showed that, in such samples, "low-protein" trehalose-water domains are present, surrounded by a protein-trehalose-water background; such finding is supported by Infrared Spectroscopy (FTIR) measurements. These domains, which do not appear in the absence of the protein and in analogous sucrose systems, preferentially incorporate the incoming water at the onset of rehydration, and disappear following large hydration. This observation suggests that, in organisms under anhydrobiosis, analogous domains could play a buffering role against the daily variations of the atmospheric moisture. The reported results are rationalized by assuming sizably different protein-matrix coupling in trehalose with respect to sucrose, analogous to the one suggested for the photosynthetic reaction centre from Rhodobacter sphaeroides (F. Francia et al., J. Am. Chem. Soc., 2008, 130, 10240-10246).

Thermal denaturation of myoglobin in water--disaccharide matrixes: relation with the glass transition of the system.

Proteins embedded in glassy saccharide systems are protected against adverse environmental conditions [Crowe et al. Annu. Rev. Physiol. 1998, 60, 73-103]. To further characterize this process, we studied the relationship between the glass transition temperature of the protein-containing saccharide system (T(g)) and the temperature of thermal denaturation of the embedded protein (T(den)). To this end, we studied by differential scanning calorimetry the thermal denaturation of ferric myoglobin in water/disaccharide mixtures containing nonreducing (trehalose, sucrose) or reducing (maltose, lactose) disaccharides. All the samples studied are, at room temperature, liquid systems whose viscosity varies from very low to very large values, depending on the water content. At a high water/saccharide mole ratio, homogeneous glass formation does not occur; regions of glass form, whose T(g) does not vary by varying the saccharide content, and the disaccharide barely affects the myoglobin denaturation temperature. At a suitably low water/saccharide mole ratio, by lowering the temperature, the systems undergo transition to the glassy state whose T(g) is determined by the water content; the Gordon-Taylor relationship between T(g) and the water/disaccharide mole ratio is obeyed; and T(den) increases by decreasing the hydration regardless of the disaccharide, such effect being entropy-driven. The presence of the protein was found to lower the T(g). Furthermore, for nonreducing disaccharides, plots of T(den) vs T(g) give linear correlations, whereas for reducing disaccharides, data exhibit an erratic behavior below a critical water/disaccharide ratio. We ascribe this behavior to the likelihood that in the latter samples, proteins have undergone Maillard reaction before thermal denaturation.

Adsorbed CO on group 10 metal fragments: a DFT study.

DFT calculations on the helicopter and cartwheel rotations of one CO molecule adsorbed at the bridge site on metal-surface fragments, characterized by two (M(8)) or three (M(14)) metal-atom layers (M = Ni, Pd, Pt) were performed by the B3LYP[LANL2DZ+6-31 g(d,p)] method, to rationalize the adsorption energetics and the steric hindrance characteristics of surface CO molecules. Potential Energy Surfaces were obtained, either fixing the C-O bond-length or allowing it to change. The behavior of the three metals, as obtained from the study of the configurational space characterizing the CO adsorption on the fragments was explained on the basis of the interaction energies involved in the different CO/M systems. The results, obtained by using the M(14) fragments and varying both the C-O and the CO/M distances, point out that the CO adsorption on the Ni fragment is stabilized by surface-configurations in which the O atom is pointing toward a metal center. At variance, C-O bond elongation and stabilization occur on Pd when the O atom is situated between two palladium atoms. The CO adsorption on Pt displays similar characteristics to those observed on the Pd systems, but with the fundamental difference caused by the destabilization of the Pt-O interactions when the O atom is situated exactly between two Pt atoms. The calculations allowed us to estimate the IR spectroscopy frequency and band-broadening of the adsorbed CO stretching by a statistic analysis on a large set of energy / bond-length computed data. Good agreement with the experimental results was obtained for all the metals, in particular concerning the frequencies. Reliable band-broadenings were also obtained for the CO/Ni and CO/Pt systems, while the lower band-broadening value for the CO/Pd system was related to the small extent of the configurational sampling space.

Role of solvent on protein-matrix coupling in MbCO embedded in water-saccharide systems: a Fourier transform infrared spectroscopy study.

Embedding protein in sugar systems of low water content enables one to investigate the protein dynamic-structure function in matrixes whose rigidity is modulated by varying the content of residual water. Accordingly, studying the dynamics and structure thermal evolution of a protein in sugar systems of different hydration constitutes a tool for disentangling solvent rigidity from temperature effects. Furthermore, studies performed using different sugars may give information on how the detailed composition of the surrounding solvent affects the internal protein dynamics and structural evolution. In this work, we compare Fourier transform infrared spectroscopy measurements (300-20 K) on MbCO embedded in trehalose, sucrose, maltose, raffinose, and glucose matrixes of different water content. At all the water contents investigated, the protein-solvent coupling was tighter in trehalose than in the other sugars, thus suggesting a molecular basis for the trehalose peculiarity. These results are in line with the observation that protein-matrix phase separation takes place in lysozyme-lactose, whereas it is absent in lysozyme-trehalose systems; indeed, these behaviors may respectively be due to the lack or presence of suitable water-mediated hydrogen-bond networks, which match the protein surface to the surroundings. The above processes might be at the basis of pattern recognition in crowded living systems; indeed, hydration shells structural and dynamic matching is first needed for successful come together of interacting biomolecules.

Light-induced protein-matrix uncoupling and protein relaxation in dry samples of trehalose-coated MbCO at room temperature.

In humid samples of trehalose-coated carboxy-myoglobin (MbCO), thermally driven conformational relaxation takes place after photodissociation of the carbon monoxide (CO) molecule at room temperature. In such samples, because of the extreme viscosity of the external matrix, photodissociated CO cannot diffuse out of the protein and explores the whole (proximal and distal side) heme pocket, experiencing averaged protein heme pocket structures, as a result of the presence of Brownian motions. At variance, in very dry samples, a lower portion of the photodissociated CO diffuses from the distal to the proximal heme pocket side probing in nonaveraged structures. We revisit here the flash photolysis data by Librizzi et al. (2002) and report on new, room temperature experiments in MbCO-trehalose samples, shortly illuminated prior the laser pulse. In dry samples, pre-illumination increased the diffusion of CO from the distal to the proximal heme pocket side, which resulted in less structure than in non-pre-illuminated samples. Such an effect, which is absent in humid samples, stems from a decoupling of the protein internal degrees of freedom from those of the external water-sugar matrix. We suggest that such a decoupling can be brought about by the continuous attempts performed by the protein during pre-illumination to undergo relaxation toward the photodissociated deoxy state. This, in turn, causes a collapse in the hydrogen bond network, which connects the protein surface to the water-sugar matrix, as reported by Cottone et al. (2002) and Giuffrida et al. (2003). In the conclusion section, we discuss the possible involvement of the processes invoked to rationalize the present data, in the function of macromolecules and interactions in living cells.

Internal dynamics and protein-matrix coupling in trehalose-coated proteins.

We review recent studies on the role played by non-liquid, water-containing matrices on the dynamics and structure of embedded proteins. Two proteins were studied, in water-trehalose matrices: a water-soluble protein (carboxy derivative of horse heart myoglobin) and a membrane protein (reaction centre from Rhodobacter sphaeroides). Several experimental techniques were used: Mossbauer spectroscopy, elastic neutron scattering, FTIR spectroscopy, CO recombination after flash photolysis in carboxy-myoglobin, kinetic optical absorption spectroscopy following pulsed and continuous photoexcitation in Q(B) containing or Q(B) deprived reaction centre from R. sphaeroides. Experimental results, together with the outcome of molecular dynamics simulations, concurred to give a picture of how water-containing matrices control the internal dynamics of the embedded proteins. This occurs, in particular, via the formation of hydrogen bond networks that anchor the protein surface to the surrounding matrix, whose stiffness increases by lowering the sample water content. In the conclusion section, we also briefly speculate on how the protein-matrix interactions observed in our samples may shed light on the protein-solvent coupling also in liquid aqueous solutions.