Single particle analysis of relaxed and activated muscle thin filaments.
The movement of tropomyosin from actin's outer to its inner domain plays a key role in sterically regulating muscle contraction. This movement, from a low Ca2+ to a Ca2+-induced position has been directly demonstrated by electron microscopy and helical reconstruction. Solution studies, however, suggest that tropomyosin oscillates dynamically between these positions at all Ca2+ levels, and that it is the position of this equilibrium that is controlled by Ca2+. Helical reconstruction reveals only the average position of tropomyosin on the filament, and not information on the local dynamics of tropomyosin in any one Ca2+ state. We have therefore used single particle analysis to analyze short filament segments to reveal local variations in tropomyosin behavior. Segments of Ca2+-free and Ca2+ treated thin filaments were sorted by cross-correlation to low and high Ca2+ models of the thin filament. Most segments from each data set produced reconstructions matching those previously obtained by helical reconstruction, showing low and high Ca2+ tropomyosin positions for low and high Ca2+ filaments. However, approximately 20% of segments from Ca2+-free filaments fitted best to the high Ca2+ model, yielding a corresponding high Ca2+ reconstruction. Conversely, approximately 20% of segments from Ca2+-treated filaments fitted best to the low Ca2+ model and produced a low Ca2+ reconstruction. Hence, tropomyosin position on actin is not fixed in either Ca2+ state. These findings provide direct structural evidence for the equilibration of tropomyosin position in both high and low Ca2+ states, and for the concept that Ca2+ controls the position of this equilibrium. This flexibility in the localization of tropomyosin may provide a means of sterically regulating contraction at low energy cost.
Pirani
Alnoor
A
Department of Physiology and Biophysics, Boston University School of Medicine, 715 Albany Street, Boston, MA 02118, USA.
Xu
Chen
C
Hatch
Victoria
V
Craig
Roger
R
Tobacman
Larry S
LS
Lehman
William
W
D000199:Actins / Q000737:chemistry / Q000502:physiology / Q000648:ultrastructure; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D007091:Image Processing, Computer-Assisted; D066298:In Vitro Techniques; D008854:Microscopy, Electron; D008958:Models, Molecular; D046912:Multiprotein Complexes; D009119:Muscle Contraction / Q000502:physiology; D009124:Muscle Proteins / Q000737:chemistry* / Q000502:physiology / Q000648:ultrastructure*; D009126:Muscle Relaxation / Q000502:physiology; D018482:Muscle, Skeletal / Q000737:chemistry; D009200:Myocardial Contraction / Q000502:physiology; D009206:Myocardium / Q000737:chemistry; D011817:Rabbits; D014335:Tropomyosin / Q000737:chemistry / Q000502:physiology / Q000648:ultrastructure; D014336:Troponin / Q000737:chemistry / Q000502:physiology / Q000648:ultrastructure
D000199:Actins; D046912:Multiprotein Complexes; D009124:Muscle Proteins; D014335:Tropomyosin; D014336:Troponin; D002118:Calcium