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 (at approximately 40 to 45 degrees C) with a maximum at 41.7 +/- 0.1 degrees C and delta H approximately 1080 +/- kcal/mol is observed for both dephospho- and phospho-myosin II. Skeletal muscle myosin unfolds with less cooperativity over a wider temperature range (approximately 40 to 60 degrees C) with delta H approximately 2500 kcal/mol. The thermal unfolding of either myosin results in a loss of approximately 70% of alpha-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 approximately 49 degrees 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 approximately 41 and approximately 48 degrees 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. Study holds ProTherm entries: 7736, 7737, 7738, 7739 Extra Details: differential scanning calorimetry; protein folding;,ATP binding; circular dichroism; myosin
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:34 p.m.
|Number of data points||8|
|Proteins||Calmodulin ; Myosin light chain kinase 2, skeletal/cardiac muscle|
|Assays/Quantities/Protocols||Experimental Assay: dHcal ; Experimental Assay: Tm|
|Libraries||Mutations for sequence A:ADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADGNGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGFISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVTMMTSK/B:KRRWKKNFIAVSAANRFKKISSSGAL|