The thermal unfolding of rabbit skeletal heavy meromyosin (HMM), myosin subfragment 1, and subfragment 2 has been studied by differential scanning calorimetry (DSC). Two distinct endotherms are observed in the DSC scan of heavy meromyosin. The first endotherm, with a Tm of 41 degrees C at pH 7.9 in 0.1 M KCl, is assigned to the unfolding of the subfragment 2 domain of HMM based on scans of isolated subfragment 2. The unfolding of the subfragment 2 domain is reversible both in the isolated form and in HMM. The unfolding of subfragment 2 in HMM can be fit as a single two-state transition with a delta Hvh and delta Hcal of 161 kcal/mol, indicating that subfragment 2 exists as a single domain in HMM. The unfolding of subfragment 2 is characterized by an extraordinarily large delta Cp of approximately 30,000 cal/(deg.mol). In the presence of nucleotides, the high-temperature HMM endotherm with a Tm of 48 degrees C shifts to higher temperature, indicating that this peak corresponds to the unfolding of the subfragment 1 domain. This assignment has been confirmed by comparison with isolated subfragment 1. The stabilizing effect of AMPPNP was significantly greater than that of ADP. The vanadate-trapped ADP species was slightly more stable than M.AMPPNP with a Tm at 58 degrees C. The unfolding of subfragment 1, both in the isolated form and in HMM, was irreversible. Only a single endotherm was noted in the DSC scans of the subfragment 1 domain of HMM and in freshly prepared subfragment 1 complexes.(ABSTRACT TRUNCATED AT 250 WORDS) Study holds ProTherm entries: 3676, 3677, 3678, 3679 Extra Details: endotherms; single two-state transition; stabilizing effect;,binding of nucleotides
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:22 p.m.
|Number of data points||12|
|Proteins||Calmodulin ; Myosin light chain kinase 2, skeletal/cardiac muscle|
|Assays/Quantities/Protocols||Experimental Assay: dHcal ; Experimental Assay: Tm ; Experimental Assay: dHvH|
|Libraries||Mutations for sequence A:ADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADGNGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGFISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVTMMTSK/B:KRRWKKNFIAVSAANRFKKISSSGAL|