Sergio Giuffrida

Molecular dynamics simulation of sucrose- and trehalose-coated carboxy-myoglobin

We performed a room temperature molecular dynamics (MD) simulation on a system containing 1 carboxy‐myoglobin (MbCO) molecule in a sucrose–water matrix of identical composition (89% [sucrose/(sucrose + water)] w/w) as for a previous trehalose–water–MbCO simulation (Cottone et al., Biophys J 2001;80:931–938). Results show that, as for trehalose, the amplitude of protein atomic mean‐square fluctuations, on the nanosecond timescale, is reduced with respect to aqueous solutions also in sucrose. A detailed comparison as a function of residue number evidences mobility differences along the protein backbone, which can be related to a different efficacy in bioprotection. Different heme pocket structures are observed in the 2 systems. The joint distribution of the magnitude of the electric field at the CO oxygen atom and of the angle between the field and the CO unit vector shows a secondary maximum in sucrose, absent in trehalose. This can explain the CO stretching band profile (A substates distribution) differences evidenced by infrared spectroscopy in sucrose‐ and trehalose‐coated MbCO (Giuffrida et al., J Phys Chem B 2004;108:15415–15421), and in particular the appearance of a further substate in sucrose. Analysis of hydrogen bonds at the protein–solvent interface shows that the fraction of water molecules shared between the protein and the sugar is lower in sucrose than in trehalose, in spite of a larger number of water molecules bound to the protein in the former system, thus indicating a lower protein–matrix coupling, as recently observed by Fourier transform infrared (FTIR) experiments (Giuffrida et al., J Phys Chem B 2004;108:15415–15421). Proteins 2005.

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.

Role of residual water hydrogen bonding in sugar/water/biomolecule systems: a possible explanation for trehalose peculiarity

We report on the set of experimental and simulative evidences which enabled us to suggest how biological structures embedded in a non-liquid water–saccharide solvent are anchored to the surrounding matrix via a hydrogen bond network. Such a network, whose rigidity increases by decreasing the sample water content, couples the degrees of freedom of the biostructure to those of the matrix and gives place to protein–saccharide–water structures (protein–solvent conformational substates). In particular, the whole set of data evidences that, while the protein–sugar interaction is well described in terms of a water entrapment hypothesis, the water replacement hypothesis better describes the sugar–membrane interaction; furthermore, it gives a hint towards the understanding of the origin of the trehalose peculiarity since the biomolecule–matrix coupling, specific to each particular sugar, always results in being the tightest for trehalose. In line with the heterogeneous dynamics in supercooled fluids and in carbohydrate glasses of different residual water contents, recent results confirm, at the single molecule level, the existence of protein–solvent conformational substates, spatially heterogeneous and interconverting, whose rigidity increases by lowering the sample hydration.

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.

IDEA: Interface dynamics and energetics algorithm

IDEA, interface dynamics and energetics algorithm, was implemented, in FORTRAN, under different operating systems to mimic dynamics and energetics of elementary events involved in interfacial processes. The code included a parallel elaboration scheme in which both the stochastic and the deterministic components, involved in the developed physical model, worked simultaneously. IDEA also embodied an optionally running VISUAL subroutine, showing the dynamic energy changes caused by the surface events, e.g., occurring at the gas‐solid interface. Monte Carlo and ordinary differential equation system subroutines were employed in a synergistic way to drive the occurrence of the elementary events and to manage the implied energy flows, respectively. Biphase processes, namely isothermal and isobaric adsorption of carbon monoxide on nickel, palladium, and platinum surfaces, were first studied to test the capability of the code in modeling real frames. On the whole, the simulated results showed that IDEA could reproduce the inner characteristics of the studied systems and predict properties not yet experimentally investigated.

Proteins in amorphous saccharide matrices: Structural and dynamical insights on bioprotection

Bioprotection by sugars, and in particular trehalose peculiarity, is a relevant topic due to the implications in several fields. The underlying mechanisms are not yet clearly elucidated, and remain the focus of current investigations. Here we revisit data obtained at our lab on binary sugar/water and ternary protein/sugar/water systems, in wide ranges of water content and temperature, in the light of the current literature. The data here discussed come from complementary techniques (Infrared Spectroscopy, Molecular Dynamics simulations, Small Angle X-ray Scattering and Calorimetry), which provided a consistent description of the bioprotection by sugars from the atomistic to the macroscopic level. We present a picture, which suggests that protein bioprotection can be explained in terms of a strong coupling of the biomolecule surface to the matrix via extended hydrogen-bond networks, whose properties are defined by all components of the systems, and are strongly dependent on water content. Furthermore, the data show how carbohydrates having similar hydrogen-bonding capabilities exhibit different efficiency in preserving biostructures.Graphical abstract

SAXS study on myoglobin embedded in amorphous saccharide matrices

We report on Small Angle X-ray Scattering (SAXS) measurements performed on samples of carboxy-myoglobin and met-myoglobin embedded in low hydrated matrices of four different saccharides (trehalose, sucrose, maltose and lactose). Results confirm the already reported occurrence of inhomogeneities, which are not peculiar of trehalose samples, but appear also in maltose and lactose, and in some cases also sucrose, being dependent on the sample hydration and on the presence of sodium dithionite. This behaviour confirms our previous interpretation about the nature of the inhomogeneities, and prompt it as a possible general behaviour for highly concentrated sugar matrices.

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.

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.