Stabilization of helical order in the thick filaments by blebbistatin: further evidence of coexisting multiple conformations of myosin.
The degree of helical order of the thick filament of mammalian skeletal muscle is highly dependent on temperature and the nature of the ligand. Previously, we showed that there was a close correlation between the conformation of the myosin heads on the surface of the thick filaments and the extent of their helical order. Helical order required the heads to be in the closed conformation. In addition, we showed that, with the same ligand bound at the active site, three conformations of myosin coexisted in equilibrium. Hitherto, however, there was no detectable helical order as measured by x-ray diffraction under the temperatures studied for myosin with MgADP and the nucleotide-free myosin, raising the possibility that the concept of multiple conformations has limited validity. In this study, blebbistatin was used to stabilize the closed conformation of myosin. The degree of helical order is substantially improved with MgATP at low temperature or with MgADP or in the absence of nucleotide. The thermodynamic parameters of the disorder<-->order transition and the characteristics of the ordered array were not significantly altered by binding blebbistatin. The simplest explanation is that the binding of blebbistatin increases the proportion of myosin in the closed conformation from being negligible to substantial. These results provide further evidence for the coexistence of multiple conformations of myosin under a wide range of conditions and for the closed conformation being directly coupled to helical order.
National Institute of Arthritis, Musculoskeletal and Skin Diseases, National Institutes of Health, Department of Health and Human Services, Bethesda, Maryland, USA.
Sengen
S
Xu
Howard D
HD
White
Gerald W
GW
Offer
Leepo C
LC
Yu
D000199:Actins / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D006576:Heterocyclic Compounds, 4 or More Rings / Q000378:metabolism*; D009218:Myosins / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D016658:Psoas Muscles / Q000737:chemistry; D011817:Rabbits; D013696:Temperature; D013816:Thermodynamics; D014961:X-Ray Diffraction
D000199:Actins; D006576:Heterocyclic Compounds, 4 or More Rings; C472645:blebbistatin; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D009218:Myosins