Study of cosolvent-induced α-chymotrypsin fibrillogenesis: Does protein surface hydrophobicity trigger early stages of aggregation reaction?

The misfolding of specific proteins is often associated with their assembly into fibrillar aggregates, commonly termed amyloid fibrils. Despite the many efforts expended to characterize amyloid formation in vitro, there is no deep knowledge about the environment (in which aggregation occurs) as well as mechanism of this type of protein aggregation. Alpha-chymotrypsin was recently driven toward amyloid aggregation by the addition of intermediate concentrations of trifluoroethanol. In the present study, approaches such as turbidimetric, thermodynamic, intrinsic fluorescence and quenching studies as well as chemical modification have been successfully used to elucidate the underlying role of hydrophobic interactions (involved in early stages of amyloid formation) in α-chymotrypsin-based experimental system. © 2009 Springer Science+Business Media, LLC

A Protein Aggregation Based Test for Screening of the Agents Affecting Thermostability of Proteins

To search for agents affecting thermal stability of proteins, a test based on the registration of protein aggregation in the regime of heating with a constant rate was used. The initial parts of the dependences of the light scattering intensity (I) on temperature (T) were analyzed using the following empiric equation: I = Kagg(T−T0)2, where Kagg is the parameter characterizing the initial rate of aggregation and T0 is a temperature at which the initial increase in the light scattering intensity is registered. The aggregation data are interpreted in the frame of the model assuming the formation of the start aggregates at the initial stages of the aggregation process. Parameter T0 corresponds to the moment of the origination of the start aggregates. The applicability of the proposed approach was demonstrated on the examples of thermal aggregation of glycogen phosphorylase b from rabbit skeletal muscles and bovine liver glutamate dehydrogenase studied in the presence of agents of different chemical nature. The elaborated approach to the study of protein aggregation may be used for rapid identification of small molecules that interact with protein targets

MECHANISM OF THERMAL AGGREGATION OF RABBIT MUSCLE GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE. BIOCHEMISTRY.

Thermal denaturation and aggregation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been studied using differential scanning calorimetry (DSC), dynamic light scattering (DLS), and analytical ultracentrifugation. The maximum of the protein thermal transition (T(m)) increased with increasing the protein concentration, suggesting that the denaturation process involves the stage of reversible dissociation of the enzyme tetramer into the oligomeric forms of lesser size. The dissociation of the enzyme tetramer was shown by sedimentation velocity at 45 degrees C. The DLS data support the mechanism of protein aggregation that involves a stage of the formation of the start aggregates followed by their sticking together. The hydrodynamic radius of the start aggregates remained constant in the temperature interval from 37 to 55 degrees C and was independent of the protein concentration (R(h,0) approximately 21 nm; 10 mM sodium phosphate, pH 7.5). A strict correlation between thermal aggregation of GAPDH registered by the increase in the light scattering intensity and protein denaturation characterized by DSC has been proved

EFFECT OF ALPHA-CRYSTALLIN ON THERMAL DENATURATION AND AGGREGATION OF RABBIT MUSCLE GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE.

The study of thermal denaturation of rabbit muscle glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the presence of alpha-crystallin by differential scanning calorimetry (DSC) showed that the position of the maximum on the DSC profile (T(max)) was shifted toward lower temperatures with increasing alpha-crystallin concentration. The diminishing GAPDH stability in the presence of alpha-crystallin has been explained assuming that heating of GAPDH induces dissociation of the tetrameric form of the enzyme into dimers interacting with alpha-crystallin. The dissociation of the enzyme tetramer was shown by sedimentation velocity at 45 degrees C. Suppression of thermal aggregation of GAPDH by alpha-crystallin was studied by dynamic light scattering under the conditions wherein temperature was elevated at a constant rate. The construction of the light scattering intensity versus the hydrodynamic radius (R(h)) plots enabled estimating the hydrodynamic radius of the start aggregates (R(h,0)). When aggregation of GAPDH was studied in the presence of alpha-crystallin, the start aggregates of lesser size were observed