Hindawi Publishing Corporation Journal of Biophysics Volume 2009, Article ID 380967, 17 pages doi:10.1155/2009/380967 Research Article Tropomyosin Period 3 Is Essential for Enhancement of Isometric Tension in Thin Filament-Reconstituted Bovine Myocardium Masataka Kawai, 1 Xiaoying Lu, 1 Sarah E. Hitchcock-DeGregori, 2 Kristen J. Stanton, 1 and Michael W. Wandling 2 1 Department of Anatomy and Cell Biology, The University of Iowa, Iowa City, IA 52242, USA 2 Department of Neuroscience and Cell Biology, Robert Wood Johnson Medical School, Piscataway, NJ 08854, USA Correspondence should be addressed to Masataka Kawai, masataka-kawai@uiowa.edu Received 23 March 2009; Revised 29 May 2009; Accepted 5 July 2009 Recommended by P. Bryant Chase Tropomyosin (Tm) consists of 7 quasiequivalent repeats known as “periods,” and its specific function may be associated with these periods. To test the hypothesis that either period 2 or 3 promotes force generation by inducing a positive allosteric effect on actin, we reconstituted the thin filament with mutant Tm in which either period 2 (Δ2Tm) or period 3 (Δ3Tm) was deleted. We then studied: isometric tension, stiffness, 6 kinetic constants, and the pCa-tension relationship. N-terminal acetylation of Tm did not cause any differences. The isometric tension in Δ2Tm remained unchanged, and was reduced to ∼60% in Δ3Tm. Although the kinetic constants underwent small changes, the occupancy of strongly attached cross-bridges was not much different. The Hill factor (cooperativity) did not differ significantly between Δ2Tm (1.79 ± 0.19) and the control (1.73 ± 0.21), or Δ3Tm (1.35 ± 0.22) and the control. In contrast, pCa 50 decreased slightly in Δ2Tm (5.11 ± 0.07), and increased significantly in Δ3Tm (5.57 ± 0.09) compared to the control (5.28 ± 0.04). These results demonstrate that, when ions are present at physiological concentrations in the muscle fiber system, period 3 (but not period 2) is essential for the positive allosteric effect that enhances the interaction between actin and myosin, and increases isometric force of each cross-bridge. Copyright © 2009 Masataka Kawai et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. 1. Introduction The tropomyosin (Tm) gene is the product of gene dupli- cation [1] and its sequence reflects the presence of a 7-fold repeat that has been postulated to relate to the binding sites for the 7 actin monomers along the length of a Tm molecule [1–3]. This molecule is a coiled-coil dimeric protein, and together with the troponin complex (Tn), it regulates thin- filament activation in striated muscles. This event is initiated by Ca 2+ binding to TnC, and enhanced by myosin binding to actin. The reactions are cooperative and allosteric, as shown in solution studies [4–6], in vitro motility assays [7–10], and in muscle fiber studies [11–14]. Analyses of the mechanism that underlies thin-filament activation have indicated a role for the 7 internal quasiequivalent repeats, called “periods,” in this activation process [15–19] as well as a role for the head- to-tail association of adjacent Tm molecules [20]. Further analysis of mutants with deletions of one or more of the periodic repeats–by solution studies, in vitro motility assays, and muscle-fiber analysis–has revealed the importance of the internal periods for allosteric regulation of actin-filament activation. Specifically, an actomyosin ATPase study and in vitro motility assays have shown that the calcium ion poorly activates actin filaments that are reconstituted with Tn plus a Tm harboring a period 3 deletion (either individually or in combination with the deletion of another period) [16, 19]. Later reports showed that the same deletions reduce the activity in the in vitro motility assay [10, 17] as well as the isometric tension [13], by ∼50%. Experiments using skinned fibers are important, because force can be measured in these preparations and the measurements can be performed in solutions that are at physiological ionic strength. Extraction of the thin filaments from strips of bovine myocardium, followed by structural and stoichiometric reconstitution of the muscle with natural or recombinant proteins, generates a suitable model for such experiments [11, 12, 21–23]. The advantage of this