Thermally Induced Unfolding of Acanthamoeba Myosin II and Skeletal Muscle Myosin: Nucleotide Effects

The thermal unfolding of monomeric Acanthamoeba myosin II and rabbit skeletal muscle myosin at pH 7.5 in 0.6 M KCl has been studied by differential scanning calorimetry (DSC) and circular dichroism, A single endotherm (∼40 to 45°C) with a maximum at 41.7 ± 0.1°C and ΔH ≍ 1080 ± 180 kcal/mol is observed for both dephospho- and phospho-myosin II, Skeletal muscle myosin unfolds with less cooperativity over a wider temperature range (∼40 to 60°C) with ΔH ≍ 2500 kcal/mol. The thermal unfolding of either myosin results in a loss of ∼70% of α-helical structures. Saturation of dephospho- or phospho-myosin II with 5′-adenylylimidodiphosphate (AMPPNP) in the presence of Mg2+ produces a second endotherm with a maximum at ∼49°C. The latter observation is attributed to a stabilization of head regions by nucleotide binding. Indeed, a purified N-terminal myosin II head fragment has been found to unfold with Tmax ∼41 and ∼48°C in the absence and presence of AMPPNP, respectively. The stabilization of the head regions is less with ADP + Pi and still smaller with ADP alone, In summary, thermally induced unfolding of myosin II is affected by nucleotide binding to heads, but not by phosphorylation or even removal of a 66-amino-acid tailpiece containing phosphorylation sites. The observed differences in the cooperativity of unfolding myosin II and skeletal muscle myosin relate to differences between rod structures and possibly also head-rod interactions.