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 stericall regulating muscle contraction. This movement, from a low Ca2+ to a Ca2+-induced electron microscopy and helical reconstruction. Solution studies, however, suggest that tropornyosin oscillates dynamically between these positions at all Ca2+ levels, and that it is the position of this equilibrium that is Ca2+. Helical reconstruction reveals only the average controlled by 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, similar to20% of segments from Ca2+-free filaments fitted best to the high Ca2+ model, yielding a corresponding high treated Ca2+ reconstruction. Conversely, similar to20% of segments from Ca. 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. (C) 2004 Elsevier Ltd. All rights reserved.