Ca2+-induced Conformational Changes of Spin-labeled g2 Chain Bound to Myosin and the Effect of Phosphorylation1

Abstract

One of the low molecular weight components of myosin, g₂, was isolated by alkali treatment of myosin and was chemically modified with a spin label reagent, 4-maleimido-2, 2, 6, 6-tetramethylpiperidinooxyl. The label on g₂ showed a rather weakly immobilized ESR spectrum and it was clearly affected by Ca²⁺; the half-maximal change was at around pCa 4. The spin-labeled g₂ was incorporated into myosin by exchange with the intrinsic g₂ of myosin in 0.6 M KSCN or 4 M LiCl. The label on g₂ became strongly immobilized on association with myosin. Under the conditions used, ESR spectral change due to Ca²⁺ occurred at two different concentration ranges, which were as low as pCa 8 and at around pCa 4. Phosphorylated g₂ was isolated from myosin after the protein kinase (EC 2.7.1.37)-catalyzed phosphorylation of myosin and it was also modified with the maleimide label. Dephosphorylation of the phosphorylated g₂ was performed using E. coli alkaline phosphatase (EC 3.1.3.1). The effects of Ca²⁺ on the ESR spectra of phosphorylated and dephosphorylated g₂ were investigated in the state associated with myosin. A change in the ESR spectrum from strongly immobilized to weakly immobilized states was observed with both g₂ chains on the addition of Ca²⁺. However, the effective concentration ranges of Ca²⁺ were quite different; around pCa 4 for the phosphorylated g₂ and around pCa 8 for the dephosphorylated g₂. The results indicate that g₂ undergoes a conformational change at physiological levels of Ca²⁺ sufficient to saturate troponin, but it does not do so after phosphorylation.