Self-Association of Phosphorylase Kinase under Molecular Crowding Conditions

Abstract

Self-association of phosphorylase kinase (PhK) has been studied using analytical ultracentrifugation and dynamic light scattering under the conditions of molecular crowding arising from the presence of high concentrations of osmolyte. Sedimentation velocity analysis shows that in accordance with the predictions of molecular crowding theory, trimethylamine N-oxide (TMAO) greatly favours self-association of PhK induced by Mg 2+ and Ca 2+ . On the contrary, proline suppresses this process, probably, due to its specific interaction with PhK. We have also established that α-crystallin, a protein possessing chaperone-like activity, counteracts the self-association of PhK under mo-lecular crowding conditions. Using dynamic light scattering we have shown that the increase in the light scattering intensity accompanying self-association of PhK is due to the formation of particles having hydrodynamic radius of hundreds of nanometers. The hydrodynamic radius of the start associates (seeds of association) was found to be approximately 80 nm. TMAO facilitates the formation of the associates of larger size whereas proline and α-crystallin suppress self-association of PhK.

Protein folding and stability in the presence of osmolytes

“…It was shown that osmolytes under conditions of molecular crowding, as a rule, promote compaction of protein structure, increase protein stability, and prevent their aggregation. Thus, overall, osmolyte function on proteins under conditions of molecular crowding is generally the same as in buffer solutions of low salinity [63,[108][109][110][111][112][113][114]. It should be noted that under the conditions of molecular crowding, structures of internally disordered proteins do not undergo significant changes [115,116], which indicates that the excluded volume effect does not play a significant role in the process of osmolyte interactions with proteins.…”

Interaction of Hsp27 with Native Phosphorylase Kinase under Crowding Conditions

“…Molecular crowding noticeably influences interactions between macromolecules as well as the rate and equilibrium of intracellular biochemical processes 14–17, 24, 25. Analyzing the effect of crowding on phosphorylase b (Ph b ) and PhK, we found that crowding affects many important reactions of key enzymes of glycogenolysis such as association, conformational transitions, interaction with allosteric ligands, denaturation, and aggregation 10, 12, 13, 18–20, 24. Many effects of crowding seem to be a result of protein association and aggregation.…”

“…Interactions of sHsps with native proteins have been poorly analyzed. Only recently we have found that under crowding conditions unfractionated α‐crystallin shifts the equilibrium between large and small associates of PhK towards the formation of small oligomers 10, 13. The unfractionated α‐crystallin is predominantly present in the form of heterogeneous high‐molecular‐mass (800–900 kDa) oligomers.…”

“…The PhK with molecular mass of 1 320 kDa4 has a complex molecular organization and consists of four subunits that form a hexadecamer (αβγδ) 4 , where the γ‐subunit possesses the catalytic activity and α‐, β‐, and δ‐subunits regulate its activity 4–7. Ca 2+ and Mg 2+ stimulate the PhK activity by inducing changes in the tertiary and quaternary structure of the molecule5–9 and also stimulate hexadecamer association 10–13…”

Physicochemical changes in phosphorylase kinase induced by its cationic activator Mg2+

“…Phosphorylase kinase (PhK) is the first protein kinase to be characterized,1 and remains one of the largest and most complex members of that superfamily (review2). The enzyme from rabbit skeletal muscle, the subject of this study and all of those listed in Table I,3–24 has a mass of 1.3 MDa and is composed of four copies each of four different subunits, (αβγδ) 4 . The γ subunit (44.7 kDa) is the catalytic kinase, whereas the α (138.4 kDa), β (125.2 kDa), and δ (16.7 kDa) subunits are regulatory.…”