Electrostatic changes in phosphorylase kinase induced by its obligatory allosteric activator Ca2+

Area of Science:

• Biochemistry
• Molecular Biology
• Enzymology

Background:

• Skeletal muscle phosphorylase kinase (PhK) is a large complex crucial for activating glycogenolysis.
• PhK requires calcium ions (Ca2+) for activation, linking muscle contraction to energy mobilization.
• The precise structural changes in PhK upon Ca2+ binding have remained unclear.

Purpose of the Study:

• To investigate the physical characteristics of PhK in the presence and absence of Ca2+.
• To understand how Ca2+ binding influences PhK's structure and stability.
• To elucidate the mechanism of Ca2+-mediated PhK activation.

Main Methods:

• Purification of hexadecameric PhK.
• Biophysical analyses including fluorescence, UV absorption, FTIR, CD spectroscopy.
• Dynamic light scattering and zeta potential measurements.
• Thermal perturbation studies at varying temperatures.

Main Results:

• Ca2+ binding caused subtle changes in PhK's tertiary and secondary structure.
• Significant alterations in surface electrostatic properties, diffusion rate, and electrophoretic mobility were observed.
• A decrease in electrostatic surface charge was noted, reducing particle mobility.
• PhK became more susceptible to thermal denaturation in the presence of Ca2+.

Conclusions:

• Ca2+ binding to PhK primarily affects its surface charge and thermal stability rather than causing major secondary structure rearrangements.
• These physicochemical changes likely contribute to the regulation of glycogenolysis.
• The findings provide new insights into the allosteric regulation of PhK by Ca2+.