Proton Nuclear‐Magnetic‐Resonance Spectroscopy of Myosin Subfragment 1 Isoenzymes

Abstract

High-resolution proton N M R spectrocopy has been used to study the solution structures of the subfragment 1 (SI) 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. (1 979) Biochemistry, 18, 4238 -42431. 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 'H-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 Sl(A1) and Sl(A2) spectra indicate that the N-terminal41 residues of the A1 light chain, rich in lysine, proline and alanine, display a high degree of segmental mobility. The difference spectrum [SI(AI)-Sl(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 intermediateslow 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 H-NMR studies referred to above. In particular, the differences noted between S l(A 1 ) and S l(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 l(A 1). Together with other affinity chromatography results [Winstanley and Trayer (1 979) Biochem. Soc. Trans. 7, 703 -7041, this is good evidence for a direct interaction between this area of S l(A 1) and actin.The mechanism of muscle contraction, as originally proposed by Huxley [I] and modified by several groups of authors [2, 31, consists of a cycle of cross-bridge detachments and attachments, which are the basis of the 'sliding filament' theory, The myosin heads form cross-bridges with actin, which are then detached by ATP. The hydrolysis of ATP produces some conformational change in myosin which is restored as mechanical energy when the heads rebind actin. There is now ample evidence that this theory is correct. There are, however, considerable differences of opinion with regard to the intermolecular and intramolecular interactions involved in the actomyosin complex, and several models have been proposed, as reviewed by Taylor [4]. X-ray diffraction and electron microscopy indicate that the cross-bridge or part of it rotates during contraction [5] and the existence of a 'swivel' and a 'hinge' have been postulated in myosin [6 -1 I...

Fluorescence energy transfer between the myosin subfragment‐1 isoenzymes and F‐actin in the absence and presence of nucleotides

“…The lack of any differences between the two isoenzymes in these fluorescence resonance energy transfer studies, even in the presence of nucleotides, suggests that the role of the alkali light chains are more subtle than these steady-state measurements can resolve. The present results are consistent with the observation that the 'H-NMR spectra of acto-Sl(A1) and acto-Sl(A2) are virtually indistinguishable [21].…”

“…Our experiments have assumed that there is enough information in the myosin head region alone to facilitate any conformational changes in S1 which occur in the acto-S1 complex on binding nucleotide. Although it has been proposed that the myosin head maintains a constant orientation during the power stroke of muscle contraction and some other part of myosin shortens [69], the demonstration of work production by actomyosin machines using only the soluble S1 fragment seem to restrict conformational changes necessary for force generation to this area of the molecule [70J Furthermore, the finding that virtually all the internal mobility of the myosin molecule is confined to the S1 region and that this is severely constrained by interaction with actin supports this argument [21,22,71].…”

“…The same abbreviations in Table 7 firms the ability of S1 to complex with regulated actin in the absence of CaZ+ [60,61]. The major difference between the two S1 isoenzymes, the N-terminal 41-residue extension of the A1 light chain, is clearly visible in the 'H-NMR spectrum of SI(A1) showing that it possesses a high degree of segmental mobility [21]. This mobility is considerably constrained by direct interaction with actin in the binary complex [20 -221, possibly suggesting that any functional differences between the isoenzymes will be expressed in the acto-S1 complex.…”

“…The second major peak eluting at higher ionic strength contained principally Sl(A2) with some contaminating SI(A1). At the outset of these studies this was further purified by chromatography on either immobilized F-actin [I91 or CMcellulose [21]. More recently the S1 isocnzymes have been fractionated on SE-cellulose.…”

“…Themethod used was essentially that of Trayer and Perry [29] except that 0.6 KCI, pH 6.8, was used as the extraction medium. Subfragment-I was prepared by chyinotryptic digestion of myosin [16] as modified in [21]. Pure SI(A1) was obtained by ion-exchange chromatography on DEAE-cellulose [16].…”