Effect of α-crystallin on thermal aggregation of glycogen phosphorylase b from rabbit skeletal muscle

Abstract

Thermal aggregation of rabbit skeletal muscle glycogen phosphorylase b (Phb) has been investigated using dynamic light scattering under conditions of a constant rate of temperature increase (1 K/min). The linear behavior of the dependence of the hydrodynamic radius on temperature for Phb aggregation is consistent with the idea that thermal aggregation of proteins proceeds in the kinetic regime wherein the rate of aggregation is limited by diffusion of the interacting particles (the regime of "diffusion-limited cluster-cluster aggregation"). In the presence of alpha-crystallin, a protein exhibiting chaperone-like activity, the dependence of the hydrodynamic radius on temperature follows the exponential law; this suggests that the aggregation process proceeds in the kinetic regime where the sticking probability for colliding particles becomes lower than unity (the regime of "reaction-limited cluster-cluster aggregation"). Based on analysis of the ratio between the light scattering intensity and the hydrodynamic radius of Phb aggregates, it has been concluded that the addition of alpha-crystallin results in formation of smaller size starting aggregates. The data on differential scanning calorimetry indicate that alpha-crystallin interacts with the intermediates of the unfolding process of the Phb molecule. The proposed scheme of thermal denaturation and aggregation of Phb includes the stage of reversible dissociation of dimers of Phb into monomers, the stage of the formation of the starting aggregates from the denatured monomers of Phb, and the stage of the sticking of the starting aggregates and higher order aggregates. Dissociation of Phb dimer into monomers at elevated temperatures has been confirmed by analytical ultracentrifugation.

Kinetics of thermal aggregation of glycogen phosphorylase b from rabbit skeletal muscle: Mechanism of protective action of α‐crystallin

“…When studying the interaction of Phb with a-crystallin by differentiation scanning calorimetry (DSC), we observed the disappearance of the peak corresponding to a-crystallin in the mixture of Phb and a-crystallin. 7 Besides, the position of the maximum of the DSC profile obtained for the mixture of Phb with a-crystallin is displaced toward the lower temperatures by 0.68C in comparison with the position of the maximum for free Phb. These data testify that a-crystallin interacts with the intermediate states of unfolding of Phb.…”

“…7,20 For light scattering measurements, the commercial instrument Photocor Complex was used (Photocor Instruments; www.photocor.com). A He-Ne laser (Coherent, model 31-2082, 632.8 nm, 10 mW) has been used as a light source.…”

“…shown that a-crystallin acting as a molecular chaperone can protect a great variety of proteins from thermal aggregation, [4][5][6][7] and this protein has been found in all tissues, including skeletal muscle. 8 The ability of a-crystallin to suppress heat-induced aggregation of other proteins is reported to be due to hydrophobic interaction with them, 9,10 and it increases when a-crystallin is heated.…”

“…5 a-Crystallin was shown to prevent the aggregation of Phb and reduce the size of formed aggregates. 7 The purpose of the present work was to study the kinetics of thermal aggregation of Phb at a fixed temperature (at 488C) in the presence and absence of a-crystallin using dynamic light scattering (DLS), analytical ultracentrifugation, and measurements of the enzymatic activity, and on the basis of obtained results to propose the molecular mechanism of the protective action of a-crystallin.…”

Mechanism of suppression of dithiothreitol-induced aggregation of bovine α-lactalbumin by α-crystallin

“…com) as described in our previous works where DLS was used for the study of the kinetics of thermal aggregation of proteins [1,4,5,24]. An HeNe laser (Coherent, USA, Model 31-2082, 632.8 nm, 10 mW) was used as the light source.…”