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

Abstract

Skeletal muscle phosphorylase kinase (PhK) is a 1.3-MDa hexadecameric complex that catalyzes the phosphorylation and activation of glycogen phosphorylase b. PhK has an absolute requirement for Ca 2+ ions, which couples the cascade activation of glycogenolysis with muscle contraction. Ca 2+ activates PhK by binding to its nondissociable calmodulin subunits; however, specific changes in the structure of the PhK complex associated with its activation by Ca 2+ have been poorly understood. We present herein the first comparative investigation of the physical characteristics of highly purified hexadecameric PhK in the absence and presence of Ca 2+ ions using a battery of biophysical probes as a function of temperature. Ca 2+ -induced differences in the tertiary and secondary structure of PhK measured by fluorescence, UV absorption, FTIR, and CD spectroscopies as low resolution probes of PhK's structure were subtle. In contrast, the surface electrostatic properties of solvent accessible charged and polar groups were altered upon the binding of Ca 2+ ions to PhK, which substantially affected both its diffusion rate and electrophoretic mobility, as measured by dynamic light scattering and zeta potential analyses, respectively. Overall, the observed physicochemical effects of Ca 2+ binding to PhK were numerous, including a decrease in its electrostatic surface charge that reduced particle mobility without inducing a large alteration in secondary structure content or hydrophobic tertiary interactions. Without exception, for all analyses in which the temperature was varied, the presence of Ca 2+ rendered the enzyme increasingly labile to thermal perturbation.Keywords: Ca 2+ ; phosphorylase kinase; secondary structure; spectroscopy; surface electrostatics; tertiary structure; zeta potential In the five decades since the discovery of skeletal muscle phosphorylase kinase (PhK), the first protein kinase to be purified and characterized (Krebs and Fischer 1956), its activity (the phosphorylation and activation of glycogen phosphorylase b) and regulation thereof have been well characterized. The kinase activity of PhK has an absolute requirement for Ca 2+ ions, which couples skeletal muscle contraction with the cascade activation of glycogenolysis to supply the short-term power demands of muscle (Shulman 2005). In contrast to the large amount of published data on the control of PhK's activity, structural information on the PhK holoenzyme complex has Reprint requests to: Gerald M. Carlson, Department of Biochemistry and Molecular Biology, Mail Stop 3030, 3901 Rainbow Blvd., Kansas City, KS 66160, USA; e-mail gcarlson@kumc.edu; fax (913) 588-7440.Abbreviations: l max , maximum fluorescence emission wavelength; ANS, 1-analinonaphthalene-8-sulfonate; CaM, calmodulin; CD, circular dichroism; cTnC, troponin C (cardiac isoform); cTnI, troponin I (cardiac isoform); d, hydrodynamic diameter; DLS, dynamic light scattering; FTIR, Fourier transform infrared; I E , fluorescence emission intensity; PDI, polydispersity index; PhK, ph...

Physicochemical changes in phosphorylase kinase associated with its activation

“…5). The most negative zeta potential was observed for Ca 2+ -free PhK at pH 6.8, and that became significantly less negative upon the binding of Ca 2+ , corroborating previous results (Priddy et al 2007). Above pH 7.6 there was little change in zeta potential for either Ca 2+ -free or Ca 2+ -bound PhK, but the least negative zeta potential was observed for the latter form at pH 8.2.…”

“…Electrostatic surface charge/zeta potential Zeta potential measurements obtained by analyzing the shear surface electric potential of colloidal particles in solution provide an estimate of effective surface charge (Hunter 1981), and this value for PhK at the fixed pH of 6.8 was recently shown to be dramatically altered by its binding of Ca 2+ (Priddy et al 2007). Using phase analysis -bound forms of PhK to become progressively less negative; however, the greatest effects of pH were observed for Ca 2+ -free PhK below pH 7.6 (Fig.…”

Structure and Location of the Regulatory β Subunits in the (αβγδ)4 Phosphorylase Kinase Complex

“…CD-Far-UV CD spectra were collected for PhK and the ␣␥␦ subcomplex using previously described conditions (34). Secondary structure content was estimated using the Dichroweb software package (35), which permits analysis of secondary structure by CONTIN, SELCON, and CDSSTR (36, 37).…”

Structural Evidence for Co-Evolution of the Regulation of Contraction and Energy Production in Skeletal Muscle

“…The possibility of a Ca 2+ -dependent electrostatic switch in γ similar to that observed in TnI is discussed in a recent report that Ca 2+ induces a large change in the surface electrostatics of the PhK complex. 34 In the genes for fast skeletal muscle TnI from quail, chicken, mouse, and human, the sequence similarity between γ and TnI (Fig. 4) occurs in a domain encoded by a single exon, which spans residues 92-150, [35][36][37] a domain that includes the entire inhibitory peptide region of TnI; no other sequence similarity is present in the two proteins outside of the domain encoded by this exon.…”