Search

Peptides corresponding to the N-terminus of skeletal myosin light chain 1 (rsMLC1 1–37) and the short loop of human cardiac β-myosin (hcM398–414) have been shown to interact with skeletal F-actin by NMR and fluorescence measurements. Skeletal tropomyosin strengthens the binding of the myosin peptides to actin but does not interact with the peptides. The binding of peptides corresponding to the inhibitory region of cardiac troponin I (e.g. hcTnI128–153) to F-actin to form a 1 : 1 molar complex is also strengthened in the presence of tropomyosin. In the presence of inhibitory peptide at relatively lower concentrations the myosin peptides and a troponin I peptide C-terminal to the inhibitory region, rcTnI161–181, all dissociate from F-actin. Structural and fluorescence evidence indicate that the troponin I inhibitory region and the myosin peptides do not bind in an identical manner to F-actin. It is concluded that the binding of the inhibitory region of troponin I to F-actin produces a conformational change in the actin monomer with the result that interaction at different locations of F-actin is impeded. These observations are interpreted to indicate that a major conformational change occurs in actin on binding to troponin I that is fundamental to the regulatory process in muscle. The data are discussed in the context of tropomyosin's ability to stabilize the actin filament and facilitate the transmission of the conformational change to actin monomers not in direct contact with troponin I. [ABSTRACT FROM AUTHOR]

The interaction between actin and caldesmon that is associated with the inhibition of actomyosin ATPase activity in smooth muscle has been studied using 1H-NMR spectroscopy. Binding studies using the intact molecules were complemented by the use of thrombic cleavage fragments of both turkey and chicken gizzard catdesmon as well as defined peptides of actin, in order to investigate the conformational properties of caldesmon and to localise regions of the primary structures that participate in protein-protein contacts. The binding of caldesmon is shown to involve distinct segments on the N-terminal region (residues 1 – 44) of actin, as previously observed for the inhibitory component of the thin filament of striated muscle, troponin I [Levine et al. (1988) Eur. J. Biochem. 153, 389 – 397]. The comparable structural properties of these tissue-specific inhibitors of actomyosin ATPase and the similarities in their mode of interaction at the N-terminal region of actin suggest common aspects to the structural mechanism for thin-filament regulation in smooth and striated muscle. Unlike the inhibitory interaction of troponin I, however, the binding of caldesmon to the N-terminal region of actin directly involves groups within residues 20 – 41 of actin that are also recognised by myosin subfragment 1. The complementary segment of caldesmon has been localised to a 15-kDa thrombic fragment (residues 483 – 578) derived from the N-terminal portion of a 35-kDa cleavage product from the C-terminal of caldesmon whose interaction with actin is modulated by calmodulin. The results are discussed in relation to the calcium-mediated mechanism for thin- filament regulation in smooth and striated muscle. [ABSTRACT FROM AUTHOR]

High-resolution proton NMR spectrocopy has been used to study the solution structures of the subfragment 1 (S1) isoenzymes (containing either the A1 or A2 light chains) from rabbit skeletal muscle myosin and to investigate their interaction with actin. Superimposed upon broad components, the narrow signals of the S1 spectra are unexpectedly sharp, indicating that domains of varying sidechain mobility occur in the conformation adopted in solution. These observations are in agreement with previous studies of the mixed isoenzymes [Highsmith et al. (1979) Biochemistry, 18, 4238 -4243]. Peptide amide exchange studies show also that the S 1 structure accommodates fluctuations of sufficient amplitude to allow most of the peptide groups to come into contact with the solvent on the time scale,of the 1H-NMR experiment. The overall impression is that S1 is a molecule possessing backbone motility as well as domains of different sidechain mobility. Close comparison of the S1(A1) and S1(A2) spectra indicate that the N-terminal 41 residues of the Al light chain, rich in lysine, proline and alanine, display a high degree of segmental mobility. The difference spectrum [S1(A1)-S1(A2)] obtained closely resembles the spectral simulation of the 41 -residue segment. Upon addition of actin, many of the narrow S1 resonances decrease in intensity or progressively disappear altogether, indicative of intermediate-slow exchange conditions consistent with the recognised high affinity between the two proteins. These changes are interpreted as an overall modulation in the observed and hence more mobile regions of S1 as has been suggested in earlier 1H-NMR studies referred to above. In particular, the differences noted between S 1(A 1) and S 1(A2) have now largely disappeared in their complexes with actin indicating a marked reduction in the segmental mobility of the N-terminal region of the light chain in S 1(A 1). Together with other affinity chromatography results [Winstanley and Trayer (1979) Biochem. Soc. Trans. 7, 703-704], this is good evidence for a direct interaction between this area of S1(A1) and actin. [ABSTRACT FROM AUTHOR]

Earlier 1H-NMR experiments on the myosin subfragment-1 (S1) light chain isoenzymes from rabbit fast muscle, containing either the A1 or the A2 alkali light chains [S1(A1) or S1(A2)], have shown that the 41-residue N-terminal extension of A1, rich in proline, alanine and lysine residues, is freely mobile in solution but that this mobility is constrained in the acto-S1(A1) complex [Prince et al. (1981) Eur. J. Biochem. 121,213–219]. It is now established that this N-terminal region of the A l-light chain interacts directly with the C-terminal region of actin in the acto-Sl (A1) complex. This was shown by covalently labelling the Cys-374 residue of actin with a spin-label and observing the enhanced relaxation this paramagnetic centre induced in the 1H-NMR spectrum of S1(A1). In particular, the signal arising from the -N+ (CH3)3 protons of α-N-trimethylalanine (Me3Ala) were monitored as this residue is uniquely sited at the N-terminus of the A1 light chain [Henry et al. (1982) FEBS Lett. 144, 11 — 15]. Experiments using complexes of actin with either the N-terminal 37-residue peptide of A1, S1 (A1) or heavy meromyosin indicate that the N-terminal region of A1 is binding in a similar manner to actin in each case, with the N-terminal Me3A1a residue within 1.5 nm of the spin label introduced to Cys-374 of actin. A similar strategy was adopted to show that the Me3Ala residue can also be found close (<1.5 rim) to the fast-reacting SH1 thiol group on the S1 heavy chain. These data, together with published work, have been used to suggest a possible organisation for the polypeptide chains in the myosin head. [ABSTRACT FROM AUTHOR]

The N terminus of skeletal myosin light chain 1 and the cardiomyopathy loop of human cardiac myosin have been shown previously to bind to actin in the presence and absence of tropomyosin (Patchell, V. B., Gallon, C. E., Hodgkin, M. A., Fattoum, A., Perry, S. V., and Levine, B. A. (2002) Eur. J. Biochem. 269, 5088–5100). We have extended this work and have shown that segments corresponding to other regions of human cardiac β-myosin, presumed to be sites of interaction with F-actin (residues 554–584, 622–646, and 633–660), likewise bind independently to actin under similar conditions. The binding to F-actin of a peptide spanning the minimal inhibitory segment of human cardiac troponin I (residues 134–147) resulted in the dissociation from F-actin of all the myosin peptides bound to it either individually or in combination. Troponin C neutralized the effect of the inhibitory peptide on the binding of the myosin peptides to F-actin. We conclude that the binding of the inhibitory region of troponin I to actin, which occurs during relaxation in muscle when the calcium concentration is low, imposes conformational changes that are propagated to different locations on the surface of actin. We suggest that the role of tropomyosin is to facilitate the transmission of structural changes along the F-actin filament so that the monomers within a structural unit are able to interact with myosin. [ABSTRACT FROM AUTHOR]