טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentKisiliak Renata
SubjectThe Mechanisms of Thermal Stabilization of Proteins by
Sugars in Aqueous Solutions
DepartmentDepartment of Biotechnology and Food Engineering
Supervisor Professor Yoav Livney
Full Thesis textFull thesis text - English Version


Abstract

Thermal stability of globular proteins is important in nature and in numerous processes in biotechnological, food and pharmaceutical applications. Sugars provide significant protection against thermal denaturation of globular proteins, but the mechanisms of this protection are still incompletely understood, particularly regarding the relationship between stereochemical structure of the sugars and their effectiveness as protein stabilizers.

Here we have used several highly sensitive techniques for following protein denaturation, in conjunction with comparison to sugar hydration numbers, to shed new light on this important and intriguing problem. We have chosen to work with isomeric sugars, which differ only stereochemically, to systematically study the effect of sugar structure on hydration-mediated effect on protein stability.

Experiments we performed by ITC have shown that there is no direct binding interaction between the protein and the sugars. This supports the hypothesis that sugars affect protein indirectly, via water. Moreover, using ITC, we have found that sugars favor the native (folded) state of a protein over the denatured (unfolded) state, and apparently induce refolding upon increasing sugar concentration.

Using differential scanning calorimetry (DSC), a sensitive microcalorimetric technique, we found increased denaturation temperature (Td) of β-lg, a model protein, with increasing sugar concentration for all sugars. The slopes (Kd) of plots of ∆Td (the change in denaturation temperature compared to the value in pure water) versus sugar concentration were significantly different between the three isomeric aldohexoses studied, in the following order: galactose> glucose>mannose. The order of the three isomeric disaccharides studied was trehalose>cellobiose>maltose, although the differences here were less significant (probably because they are all dimers of glucose). Those findings indicate that different orientations of sugars hydroxyl groups impose different perturbations of water hydrogen bonds around them. We propose that the better a template a sugar isomer is for cooperative hydration, the larger the hydration layer of the sugar, and the worse a co-solvent it is (entropically) for the protein. Therefore, the better the sugar’s protective effect on the protein, as it favors its compact globular conformation.

The above results were supported by spectroscopic studies (FTIR) combined with the chemometric technique based on Partial Least Squares (PLS) analysis. Quantitative analysis of the protein secondary structure showed that sugars hinder the changes in protein conformation at high temperatures.

Our main finding is that the values of Kd which reflects the “strength” of sugar thermal protection on the protein, correlated with the hydration numbers of the sugars within each group of isomers.