In this thesis a study, both experimental and computational, on saccharide–based biopreservation is presented, with a particular focus on the role of water in the process. Experiments and simulations have been performed on model systems constituted by a protein (myoglobin) embedded in amorphous solid saccharide–water matrices, which may contain also cosolutes to alter their properties. This study has a dual aim: (a) The understanding of the role of the hydrogen–bond (HB) network present in the saccharide matrix, and its modifications induced by solute content and nature, in the process of biopreservation. Many, among the hypotheses currently discussed to explain the effectiveness of sugars on preservation of biomolecules, attribute a outstanding relevance to water present in the matrix and its relation with the sugar. The hypothesis here supported is that the HB network, its strength and its relation with the embedded biomolecules, plays the leading role in the biopreservation mechanism. The different properties of the HB networks generated by different saccharides would be also at the basis of the peculiar efficiency of trehalose with respect to the other sugars. To this aim, experiments have been performed with variable water content and protein–sugar ratio, as well as by including cosolutes able to perturb the HB network properties in the system. Particular attention has been paid in the evaluation of the matrix properties along with those of the embedded protein. (b) The deepening of the knowledge on the mechanism of protein preservation, in particular with respect to trehalose efficiency, by a combined set of studies, which explore different spatial and temporal scales. Many results have been reported in the literature for model systems studied with a lot of techniques. This resulted in a bunch of hypotheses, of which many are likely to hold true only in the systems they have been formulated. At variance,adopting a multi–technique approach would enable to draw a consistent picture of trehalose biopreservation process. From an experimental point of view, the study was performed with Fourier–Transform Infrared Spectroscopy (FTIR) and Small Angle X–Ray Scattering (SAXS). FTIR gives information about the structure of the sample at atomistic level and on the strength of intermolecular interactions, as probed by alteration of molecular vibration frequencies. Classical FTIR spectroscopy is not generally used for the study of protein dynamics. Here, the dynamics information is conveyed by the alteration of band shapes and position upon perturbation of the sample. As such, it can be studied by analysing the behaviour of infrared bands as a function of temperature. FTIR measurements were performed in trehalose–water and myoglobin–trehalose–water systems containing various cosolutes with the aim to characterise how they alter the relative populations of different classes of water molecules present in the samples, hence how the HB network is influenced. The knowledge acquired from the study of cosolute–containing trehalose samples has been applied to study the behaviour of other saccharide matrices (sucrose, maltose, lactose, raffinose) both in the presence and in the absence of proteins and at different protein sugar ratios. FTIR measurements as a function of temperature has been performed to complete previous studies and to allow a better comparison with simulative and experimental data on analogous systems present in literature. Classical, state of the art, Molecular Dynamics (MD) simulation have been performed on binary trehalose–water and ternary myoglobin–trehalose–water systems at different temperatures, in the range 50-400 K. A deeper understanding of the different roles of sugar and water molecules is obtained by comparing the outcomes of simulations of the above ternary systems with those of the same systems in which the dynamics of one of the components (water) has been constrained. This has been made either by blocking both the translational and the rotational motions of the molecules, or by blocking only translations, while allowing the water molecule to rotate, or by restraining the water motions by means of a harmonic potential. The results from MD and FTIR allow to study the relation between protein and matrix structure and dynamics at molecular level. However these techniques are not really suitable to obtain information on larger structural alterations and in particular, because of their spatial scale, they are not able to mark the presence of micro- or mesoscopic inhomogeneities. We therefore performed SAXS measurements on binary (saccharide-water) and ternary (MbCO saccharide-water) systems with the aim to detect the effects of the embedded protein on a larger spatial scale. SAXS was chosen since it is a most suitable technique to investigate micro-nano structures having electronic densities that differ from their surrounding. This thesis is structured as follow. After a general introduction on the preservation of biological molecules and on the actual state of affairs with biopreservation by saccharides (chapter 2), a presentation on the chemical and biological properties of the biomolecules utilised is given (chapter 3), which includes also a discussion on the effects of cosolutes on solutions and hydrated matrices containing biological structures (Hofmeister effects, section 3.3). The experimental and simulative techniques are presented (chapter 4), and then the results for each technique are shown and discussed (chapters 5 for FTIR, 6 for MD and 7 for SAXS). Chapter 8 is a global discussion on the results, where also some general conclusions are drawn.

Giuffrida, . (2014). ROLE OF WATER IN SACCHARIDE BASED BIOPRESERVATION.

ROLE OF WATER IN SACCHARIDE BASED BIOPRESERVATION

GIUFFRIDA, Sergio
2014-04-14

Abstract

In this thesis a study, both experimental and computational, on saccharide–based biopreservation is presented, with a particular focus on the role of water in the process. Experiments and simulations have been performed on model systems constituted by a protein (myoglobin) embedded in amorphous solid saccharide–water matrices, which may contain also cosolutes to alter their properties. This study has a dual aim: (a) The understanding of the role of the hydrogen–bond (HB) network present in the saccharide matrix, and its modifications induced by solute content and nature, in the process of biopreservation. Many, among the hypotheses currently discussed to explain the effectiveness of sugars on preservation of biomolecules, attribute a outstanding relevance to water present in the matrix and its relation with the sugar. The hypothesis here supported is that the HB network, its strength and its relation with the embedded biomolecules, plays the leading role in the biopreservation mechanism. The different properties of the HB networks generated by different saccharides would be also at the basis of the peculiar efficiency of trehalose with respect to the other sugars. To this aim, experiments have been performed with variable water content and protein–sugar ratio, as well as by including cosolutes able to perturb the HB network properties in the system. Particular attention has been paid in the evaluation of the matrix properties along with those of the embedded protein. (b) The deepening of the knowledge on the mechanism of protein preservation, in particular with respect to trehalose efficiency, by a combined set of studies, which explore different spatial and temporal scales. Many results have been reported in the literature for model systems studied with a lot of techniques. This resulted in a bunch of hypotheses, of which many are likely to hold true only in the systems they have been formulated. At variance,adopting a multi–technique approach would enable to draw a consistent picture of trehalose biopreservation process. From an experimental point of view, the study was performed with Fourier–Transform Infrared Spectroscopy (FTIR) and Small Angle X–Ray Scattering (SAXS). FTIR gives information about the structure of the sample at atomistic level and on the strength of intermolecular interactions, as probed by alteration of molecular vibration frequencies. Classical FTIR spectroscopy is not generally used for the study of protein dynamics. Here, the dynamics information is conveyed by the alteration of band shapes and position upon perturbation of the sample. As such, it can be studied by analysing the behaviour of infrared bands as a function of temperature. FTIR measurements were performed in trehalose–water and myoglobin–trehalose–water systems containing various cosolutes with the aim to characterise how they alter the relative populations of different classes of water molecules present in the samples, hence how the HB network is influenced. The knowledge acquired from the study of cosolute–containing trehalose samples has been applied to study the behaviour of other saccharide matrices (sucrose, maltose, lactose, raffinose) both in the presence and in the absence of proteins and at different protein sugar ratios. FTIR measurements as a function of temperature has been performed to complete previous studies and to allow a better comparison with simulative and experimental data on analogous systems present in literature. Classical, state of the art, Molecular Dynamics (MD) simulation have been performed on binary trehalose–water and ternary myoglobin–trehalose–water systems at different temperatures, in the range 50-400 K. A deeper understanding of the different roles of sugar and water molecules is obtained by comparing the outcomes of simulations of the above ternary systems with those of the same systems in which the dynamics of one of the components (water) has been constrained. This has been made either by blocking both the translational and the rotational motions of the molecules, or by blocking only translations, while allowing the water molecule to rotate, or by restraining the water motions by means of a harmonic potential. The results from MD and FTIR allow to study the relation between protein and matrix structure and dynamics at molecular level. However these techniques are not really suitable to obtain information on larger structural alterations and in particular, because of their spatial scale, they are not able to mark the presence of micro- or mesoscopic inhomogeneities. We therefore performed SAXS measurements on binary (saccharide-water) and ternary (MbCO saccharide-water) systems with the aim to detect the effects of the embedded protein on a larger spatial scale. SAXS was chosen since it is a most suitable technique to investigate micro-nano structures having electronic densities that differ from their surrounding. This thesis is structured as follow. After a general introduction on the preservation of biological molecules and on the actual state of affairs with biopreservation by saccharides (chapter 2), a presentation on the chemical and biological properties of the biomolecules utilised is given (chapter 3), which includes also a discussion on the effects of cosolutes on solutions and hydrated matrices containing biological structures (Hofmeister effects, section 3.3). The experimental and simulative techniques are presented (chapter 4), and then the results for each technique are shown and discussed (chapters 5 for FTIR, 6 for MD and 7 for SAXS). Chapter 8 is a global discussion on the results, where also some general conclusions are drawn.
14-apr-2014
BIOPRESERVATION
Giuffrida, . (2014). ROLE OF WATER IN SACCHARIDE BASED BIOPRESERVATION.
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10447/91324
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