Differential scanning calorimetric study of the thermal unfolding of the motor domain fragments of Dictyostelium discoideum myosin II.
The thermal unfolding of two recombinant fragments of the head of Dictyostelium discoideum myosin II was studied by differential scanning calorimetry. These fragments M754 and M761 correspond to the globular motor portion of the myosin head that contains ATP- and actin-binding sites but lacks the light chain binding domain. Our results show that M754 is less thermostable than M761: the maximum of the thermal transition occurred at 41.7 degrees C for M754 and at 45.6 degrees C for M761, and the calorimetric enthalpy value determined for M754 (677 kJ/mol) was about half of that for M761 (1417 kJ/mol). This indicates that the region containing residues 755-761 plays a very important role in the structural stabilization of the entire globular motor part of the myosin head. ADP binding induces structural changes in both myosin fragments which are reflected in a 2-3.5 degrees C shift of the thermal transitions to higher temperature. The formation of stable ternary complexes of these myosin fragments with ADP and phosphate analogues such as orthovanadate, beryllium fluoride or aluminium fluoride causes additional structural changes which are reflected in a pronounced increase of thermal stability. The effect of beryllium fluoride was less distinct than that of aluminium fluoride or orthovanadate. In general, the changes caused by various phosphate analogues were similar to those observed with skeletal myosin subfragment 1. Thus, structural changes revealed by differential scanning calorimetry in the myosin head, that are due to the formation of stable ternary complexes with ADP and Pi analogues, occur mainly in the globular motor portion of the head.
Levitsky
D I
DI
A. N. Bakh Institute of Biochemistry, Russian Academy of Sciences, Moscow.
Ponomarev
M A
MA
Geeves
M A
MA
Shnyrov
V L
VL
Manstein
D J
DJ
D000199:Actins / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D002152:Calorimetry, Differential Scanning; D004023:Dictyostelium / Q000737:chemistry*; D015879:Myosin Subfragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009218:Myosins / Q000737:chemistry* / Q000235:genetics; D017510:Protein Folding
D000199:Actins; D015879:Myosin Subfragments; D000244:Adenosine Diphosphate; D009218:Myosins