Differential scanning calorimetry of the unfolding of myosin subfragment 1, subfragment 2, and heavy meromyosin.
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)
Shriver
J W
JW
Department of Medical Biochemistry, School of Medicine, Southern Illinois University, Carbondale 62901.
Kamath
U
U
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002152:Calorimetry, Differential Scanning; D009132:Muscles / Q000032:analysis / Q000187:drug effects; D015879:Myosin Subfragments / Q000032:analysis; D011487:Protein Conformation; D011817:Rabbits; D013816:Thermodynamics
D015879:Myosin Subfragments; D000255:Adenosine Triphosphate