The N-terminal region in actin has been shown to interact with both myosin and troponin (Tn) during the cross-bridge cycle and in regulation. To study the role of this region in regulation, we used yeast actin mutants with increased and decreased numbers of acidic residues. The mutants included D24A/D25A, with Asp(24) and Asp(25) replaced with alanines; DNEQ, with the substitution of Asp(2) and Glu(4) with their amide analogs; and 4Ac, with Glu(3) and Asp(4) inserted in lieu of Ser(3). In the in vitro motility assay, using reconstituted regulated thin filaments, the sliding speeds of DNEQ, D24A/D25A, and 4Ac were similar at all pCa values. Thus, Ca(2+)-sensitivity of the thin filaments and the inhibitory function of TnI appear to be insensitive to changes in charge (+/-2) at the N-terminus of actin, suggesting little, if any, role of that actin region in regulation. A Ca(2+)-independent conformational change in that region was detected upon troponin binding to actin-Tm via an increase in the fluorescence of a pyrene probe attached to another yeast actin mutant that we used (Cys(1)).

The contraction of vertebrate striated muscle is regulated by the thin filament-associated proteins tropomyosin (Tm) 1 and troponin (Tn), which modulate the interaction of actin and myosin in a Ca 2ϩ -dependent fashion (2). Each Tm molecule associates with one Tn molecule and seven actin monomers. The amino acid sequence of Tm contains a pattern of charged and uncharged amino acids that repeats 14 times along its length (3). As each pair of repeats corresponds to an actin monomer along the actin filament, it has been inferred that the binding of Tm to actin is dominated by electrostatic interactions (1).Tn is composed of three subunits: TnT, TnI, and TnC. The TnC subunit binds Ca 2ϩ and confers calcium sensitivity to the actin-Tm-Tn system. According to the three-state model of thin filament regulation (4), the actin-Tm-Tn complex can assume three structural states: blocked, closed, and open. In the blocked state there is a very low incidence of myosin binding. When Ca 2ϩ binds to TnC, the Tm-Tn complex shifts to the closed state, uncovering additional myosin weak binding sites on the actin filament. The increase in weak binding and the initial strong binding of myosin induce Tm-Tn to shift from the closed (where it prevents myosin strong binding) to the open state. According to this model, the azimuthal shift of Tm-Tn around the axis of the actin filament sterically regulates myosin binding to actin.Despite the elegance of the three-state model, it cannot explain well the findings of earlier acto-S1 ATPase solution studies (5-7). Results from these studies describe the ability of Tm alone to both inhibit, at low S1 concentrations, actin-activated S1 ATPase rates and to potentiate the reaction at intermediate, nonsaturating concentrations of S1. Although inhibition can readily be explained using the steric block model, the potentiation suggests the presence of an allosteric component in actomyosin regulation.The Ca 2ϩ -induced Tm-Tn movement on actin was first indicated by x-ray diffraction studies (8 -10). Electron microscopy has also been used to directly visualize this shift in Tm-Tn position (11)(12)(13)(14)(15)(16), and the studies of Limulus muscle (17, 18) and vertebrate muscle (19) identified the positions for the Tm-Tn complex on actin in the presence and absence of Ca 2ϩ . A high resolution model of the Tm-F-actin complex was proposed by Lorenz et al. (1) on the basis of their x-ray fiber diffraction investigation. According to these authors, Tm alone and Tm-Tn in the presence of Ca 2ϩ reside in the same closedstate orientation on the actin filament. In this study Lorenz et al. (1) predict that 16 actin residues participate in the electrostatic interactions between F-actin and Tm and calculate their ⌬G contributions to this interaction. Our choice of residue 311 as a suitable starting point to test the predictions of this model and to gain additional insight into Tm regulatory function was based on two criteria. First, according to Lorenz et al. (1) residue 311 has a relatively high energy contrib...

No abstract

Vertebrate striated muscle contraction is regulated in a Ca 2؉ -dependent fashion by tropomyosin (Tm) and troponin (Tn). This regulation involves shifts in the position of Tm and Tn on actin filaments and may include conformational changes in actin that are then communicated to myosin subfragment 1 (S1). To determine whether subdomain 2 of actin plays a role in this regulation, the DNase-I loop 38 -52 of this subdomain was cleaved by subtilisin between residues Met 47 and Gly 48 . Despite impaired unregulated function, the potentiation and regulation of cleaved actin movement in the in vitro motility assay was not significantly different from that of uncleaved actin. Stopped-flow measurements of ADP release from regulated and unregulated cleaved acto-S1 showed a marked increase in ADP release from acto-S1 in the presence of the regulatory complex. The enhancement of the actin affinity for S1 in the presence of regulatory proteins was greater for uncleaved than for cleaved F-actin. Finally, both cleaved and uncleaved actins protect myosin loop 1 from papain cleavage equally well. Our results suggest that the potentiation of actin function in the in vitro motility assay by regulatory proteins stems from changes in cross-bridge cycle kinetics. In addition, the unimpaired calcium-sensitive regulation of cleaved actin indicates that subdomain 2 conformation does not play an essential role in the regulation process.