ETD

Archivio digitale delle tesi discusse presso l'Università di Pisa

Tesi etd-11222016-081326


Tipo di tesi
Tesi di laurea magistrale
Autore
PACHETTI, MARIA
URN
etd-11222016-081326
Titolo
Dynamics and stability of proteins embedded in glass-forming matrices
Dipartimento
FISICA
Corso di studi
FISICA
Relatori
relatore Prof. Capaccioli, Simone
Parole chiave
  • stability
  • proteins
  • IR
  • glass transition
  • dynamics
  • DSC
  • BDS
Data inizio appello
12/12/2016
Consultabilità
Non consultabile
Data di rilascio
12/12/2025
Riassunto
Proteins are three-dimensional bio-polymers characterized by complex dynamics and structure. Both local (side-chain groups) and conformational dynamics are involved in the biological functions as well as in the mechanisms of denaturation and degradation. It has been proved that both the slow conformational and the fast local protein dynamics in non-aqueous solvents are strongly affected by the properties and the amount of solvent itself, so that a sort of “slaved dynamics” of the protein has been proposed [1]. At room temperature, the amplitude and the rate of these motions, quite large in aqueous solutions, are suppressed when proteins are embedded in very viscous or glassy matrices (such as in sugars). Moreover, proteins embedded in glassy matrices show a much higher unfolding temperature compared to the case when they are solvated in aqueous media.
The topic of this thesis, i.e. the dynamics and stability of proteins, has strong applicative implications in several fields, such as biomedical, pharmaceutical, cosmetic, food industry, and so on. In particular, some reasons recommend the dispersion of proteins in glass-forming matrices. First of all, the use of glassy sugar matrices improves the conservation of the products at room temperature because the global dynamics (conformational and cooperative motions) of the system is slowed down. Other important implications are linked to the cryoprotection at low temperature. With respect to aqueous solutions, a glass-forming matrix is favored to cool down the system at low temperature, since it avoids the crystallization of the protein environment, that can cause harmful effects like local stresses and solute precipitation. Finally, for high temperature preservation, glassy solvents are fundamental to slow down the conformational motions involved in the unfolding process, namely the loss of the native-like secondary structure of protein. Despite the large amount of studies reported in literature on these systems, only some partial aspects have been highlighted so far: for example, the effect of the kind of solvents on the modification of the secondary structure, and the effects of these matrices on the fast dynamics (ns-ps) of various peptides, visible for instance with neutron scattering. Moreover, the majority of the experimental studies performed up to now has been concentrated on proteins in water solutions or in aqueous mixtures with other organic solvents. What is still needed is a systematic study of the combined effect of glass-forming matrices on the slow dynamics and on the stability of the proteins.
In this thesis project some freeze-dried (lyophilized) mixtures of a globular protein (hen egg withe lysozyme) and glass-forming solvents (glycerol, sorbitol, glucose, trehalose, sucrose, levoglucosan), characterized by different viscosity and molecular complexity, have been analyzed with the following techniques: Differential Scanning Calorimetry (DSC), Broadband Dielectric Spectroscopy (BDS) and Infrared Spectroscopy (IR), each of them directed to monitor different observables and phenomena.
DSC has allowed to observe, first, the release of cooperative degrees of freedom at the glass transition temperature, manifesting itself in a step-like trend of the specific heat and, secondly, the denaturation peak, linked to protein unfolding, occurring at high temperatures. Both these temperatures have been observed to change along with the viscosity of the solvent. Additionally, a linear correlation was found between the unfolding temperature and the glass transition temperature of the mixtures.
BDS is a linear response technique able to observe the reorientational molecular dynamics of the permanent dipoles present in the mixtures, by means of the relaxation function of the polarization. The study has been performed in a timescale range from 10 ks to 10 ns, above and below the glass transition of the mixtures. In literature this kind of experiments is usually performed on proteins in water solutions, but only very seldom for proteins in glassy matrices [2]. BDS spectra of this study have revealed a very rich dynamic scenario, where the slow conformational and cooperative motions of the protein mixtures are strongly affected by the solvent dynamics. On the other hand, the local motions of side-chain peptide groups remain active in the glassy state, being insensitive to the solvent dynamics. This is an important result because the current literature endorses the concept of protein dynamics slaved by the solvent [1]. If this fact is true for conformational motions, it is not so for the local ones: therefore protein dynamics is not totally frozen and suppressed in the glassy state, some activities survive. It can be so concluded that the best bio-protectors are not those having higher glass transition temperature, but those able to slow down the local modes [3].
The second phenomenon of interest for this study was the unfolding. A deeper insight on this phenomenon has been obtained by IR spectroscopy in both the Far-Infrared (FIR) and Mid-Infrared (MIR) regions, aiming to study the vibrational dynamics of the systems. This opportunity is born from a collaboration with Elettra Synchrotron@Trieste (beamline SISSI), using synchrotron IR radiation. The peculiarity of this light source is the high brightness compared to the laboratory sources in the regions investigated, and also a higher power (in FIR), increasing the signal-to-noise ratio and allowing good measurements also below 100 cm-1. Through absorption in FIR region the collective vibrational bands of the protein have been observed, and their variation with the solvent nature and the thermal treatment. This region has acquired a great importance because it allows to monitor the aggregate fibrillary states of proteins, at the base of neurodegenerative diseases [4]. The Mid-Infrared region is even more sensitive to the secondary structure. In fact, the region of the amide bands (1500-1700 cm-1) reflects the vibrational dynamics of the peptide groups responsible for the arrangements of the protein backbone: variations of intensity, position and width of such bands reflect the change in the secondary structure and give information on how the solvent acts to prevent damages during the lyophilization process and the heating treatment. Actually, the presence of a glassy solvent has been observed to slow down the unfolding kinetics, thus stabilizing the native-like secondary structure, but the extent of stabilization is linked to the degree of protection of the solvent during the freeze-drying procedure.
The present study has been carried out, by infrared and dielectric techniques, on lyophilized thin films. It could be extended to non-destructive analysis of biological samples and improved to study dynamics and stability with spatial resolution at the nanoscale level, for example using Infrared Scanning Probe (SNOM-IR, AFM-IR) and Local Dielectric Spectroscopy.

References
[1] P. W. Fenimore, H. Frauenfelder, B.H. McMahon, R. D. Young, Proc. Natl. Acad. Sci. USA 101, 14408 (2004).
[2] S. Pawlus, S. Khodadadi and A.P. Sokolov, Phys. Rev. Lett. 100, 108103 (2008).
[3] M.T. Cicerone and J.F. Douglas, Soft Matter 8, 2983 (2012).
[4] G.M. Png, R.J. Falconer, and D. Abbott, IEEE Trans. THz Sci. Technol., 6, 45-53 (2016).
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