Analysis of Sugar Bioprotective Mechanisms on the Thermal Denaturation of Lysozyme from Raman Scattering and Differential Scanning Calorimetry Investigations

Abstract

Sugar-induced thermostabilization of lysozyme was analyzed by Raman scattering and modulated differential scanning calorimetry investigations, for three disaccharides (maltose, sucrose, and trehalose) characterized by the same chemical formula (C(12)H(22)O(11)). This study shows that trehalose is the most effective in stabilizing the folded secondary structure of the protein. The influence of sugars on the mechanism of thermal denaturation was carefully investigated by Raman scattering experiments carried out both in the low-frequency range and in the amide I band region. It was determined that the thermal stability of the hydrogen-bond network of water, highly dependent on the presence of sugars, contributes to the stabilization of the native tertiary structure and inhibits the first stage of denaturation, that is, the transformation of the tertiary structure into a highly flexible state with intact secondary structure. It was found that trehalose exhibits exceptional capabilities to distort the tetra-bonded hydrogen-bond network of water and to strengthen intermolecular O-H interactions responsible for the stability of the tertiary structure. Trehalose was also observed to be the best stabilizer of the folded secondary structure, in the transient tertiary structure, leading to a high-temperature shift of the unfolding process (the second stage of denaturation). This was interpreted from the consideration that the transient tertiary structure is less flexible and inhibits the solvent accessibility around the hydrophobic groups of lysozyme.

Self-assembly of trehalose molecules on a lysozyme surface: the broken glass hypothesis

“…46 The sugar effect becomes apparent starting from low sugar weight concentrations of as little as 20 %. Both molecular dynamics 15 and experimental 47 results demonstrate the reduced fluctuations of the lysozyme in trehalose solutions.…”

Slowing down of water dynamics in disaccharide aqueous solutions

“…The back-scattering Raman spectra were recorded in the 10-300 cm -1 spectral window using a Dilor-XY spectrometer equipped with a liquid nitrogen cooled charge-coupled-device detector. The scattered low-frequency intensity was transformed into Raman susceptibility χ" using a procedure detailed in previous studies [23]. χ"(ν) is related to the VDOS g(ν) by the relation…”

“…Indeed, sugars form numerous hydrogen bonds (HBs) with water [18,19], whose dynamics is strongly slow down in their hydration shell [15,20,21,22]. Recently, disaccharides were shown to decrease the flexibility of lysozyme [17,23], thereby reducing its conformational entropy. This result fully agrees with the stabilization of the tertiary structure of lysozyme at high temperatures observed in Raman scattering experiments [23] and would explain why the secondary structure of lysozyme unfolds at higher temperatures in presence of sugars [23,24].…”

“…Recently, disaccharides were shown to decrease the flexibility of lysozyme [17,23], thereby reducing its conformational entropy. This result fully agrees with the stabilization of the tertiary structure of lysozyme at high temperatures observed in Raman scattering experiments [23] and would explain why the secondary structure of lysozyme unfolds at higher temperatures in presence of sugars [23,24]. Furthermore, trehalose exhibited superior protecting capabilities [23,24] that would partially stem from its higher ability to slow down water dynamics [25], mainly because of its larger hydration number [18,26,27] .…”

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

“…Raman scattering experiments were performed also on protein-sugar solutions [14][15][16][17][18][19]. By comparing trehalose, sucrose and maltose systems, it was found out how trehalose is the most effective in stabilizing the folded secondary structure of the protein [14].…”