Interaction of isozymes of myosin subfragment 1 with actin: effect of ionic strength and nucleotide

Chalovich, Joseph M.; Stein, Leonard A.; Greene, Lois E.; Eisenberg, Evan

doi:10.1021/bi00316a011

Cited by 67 publications

(53 citation statements)

References 27 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…K d measured by cosedimentation (Fig. S1B) confirmed the much greater actin affinity of S1A1 compared with S1A2 (Table 1), consistent with previous reports for unlabeled proteins (31).…”

Section: Resultssupporting

confidence: 91%

Amplitude of the actomyosin power stroke depends strongly on the isoform of the myosin essential light chain

Guhathakurta

Próchniewicz

Thomas

2015

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

We have used time-resolved fluorescence resonance energy transfer (TR-FRET) to determine the role of myosin essential light chains (ELCs) in structural transitions within the actomyosin complex. Skeletal muscle myosins have two ELC isoforms, A1 and A2, which differ by an additional 40-45 residues at the N terminus of A1, and subfragment 1 (S1) containing A1 (S1A1) has higher catalytic efficiency and higher affinity for actin than S1A2. ELC's location at the junction between the catalytic and light-chain domains gives it the potential to play a central role in the force-generating power stroke. Therefore, we measured site-directed TR-FRET between a donor on actin and an acceptor near the C terminus of ELC, detecting directly the rotation of the light-chain domain (lever arm) relative to actin (power stroke), induced by the interaction of ATPbound myosin with actin. TR-FRET resolved the weakly bound (W) and strongly bound (S) states of actomyosin during the W-to-S transition (power stroke). We found that the W states are essentially the same for the two isoenzymes, but the S states are quite different, indicating a much larger movement of S1A1. FRET from actin to a probe on the N-terminal extension of A1 showed close proximity to actin. We conclude that the N-terminal extension of A1-ELC modulates the W-to-S structural transition of acto-S1, so that the light-chain domain undergoes a much larger power stroke in S1A1 than in S1A2. These results have profound implications for understanding the contractile function of actomyosin, as needed in therapeutic design for muscle disorders.muscle | actomyosin | light chains | fluorescence resonance energy transfer A central challenge in the biophysics of motility is to understand the molecular mechanisms of structural transitions in the actomyosin ATPase (enzyme code 3.6.4.1) cycle, in which myosin alters its affinity for actin in a nucleotide-dependent manner from weak (W) actin-binding states (with ATP or ADP-P i bound to the myosin active site) to strong (S) actin-binding states (with ADP or no nucleotide, i.e., rigor), resulting in the generation of force. It is particularly important to obtain direct information about the structural dynamics of the whole actomyosin complex, because there is evidence that each protein, when studied separately, undergoes changes in structure and dynamics during transitions from the W to S states (1-5). S states are relatively stable and easy to study, but the W actomyosin complex is less accessible to study, due to its transient and structurally dynamic nature.The myosin heavy chain starts with the N-terminal catalytic domain (CD), which contains the nucleotide-binding pocket and the actin-binding site. A small converter region connects the CD to an extended α-helical segment that forms the core of the lightchain domain (LCD) (6, 7). The LCD contains two IQ motifs that serve as binding sites for one essential light chain (ELC) and one regulatory light chain (RLC) and is proposed to function as a lever arm that amplifies small structural c...

show abstract

“…K d measured by cosedimentation (Fig. S1B) confirmed the much greater actin affinity of S1A1 compared with S1A2 (Table 1), consistent with previous reports for unlabeled proteins (31).…”

Section: Resultssupporting

confidence: 91%

Amplitude of the actomyosin power stroke depends strongly on the isoform of the myosin essential light chain

Guhathakurta

Próchniewicz

Thomas

2015

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…The observation of XB order in contraction is consistent with earlier work (7). The difference in polarization values of XBs of relaxed K104E and WT myofibrils was significant, perhaps because some myosin-actin interaction occurs at relatively low ionic strength used here (8). It was surprising to find that FWHM values for both types of myofibrils were not wider in relaxation compared with rigor.…”

Section: Discussionsupporting

confidence: 89%

Effect of a myosin regulatory light chain mutation K104E on actin-myosin interactions

Duggal

Nagwekar

Rich

et al. 2015

American Journal of Physiology-Heart and Circulatory Physiology

View full text Add to dashboard Cite

J. Effect of a myosin regulatory light chain mutation K104E on actin-myosin interactions. Am J Physiol Heart Circ Physiol 308: H1248 -H1257, 2015. First published March 13, 2015; doi:10.1152/ajpheart.00834.2014 is the most common cause of sudden cardiac death in young individuals. Molecular mechanisms underlying this disorder are largely unknown; this study aims at revealing how disruptions in actin-myosin interactions can play a role in this disorder. Cross-bridge (XB) kinetics and the degree of order were examined in contracting myofibrils from the ex vivo left ventricles of transgenic (Tg) mice expressing FHC regulatory light chain (RLC) mutation K104E. Because the degree of order and the kinetics are best studied when an individual XB makes a significant contribution to the overall signal, the number of observed XBs in an ex vivo ventricle was minimized to ϳ20. Autofluorescence and photobleaching were minimized by labeling the myosin lever arm with a relatively long-lived red-emitting dye containing a chromophore system encapsulated in a cyclic macromolecule. Mutated XBs were significantly better ordered during steady-state contraction and during rigor, but the mutation had no effect on the degree of order in relaxed myofibrils. The K104E mutation increased the rate of XB binding to thin filaments and the rate of execution of the power stroke. The stopped-flow experiments revealed a significantly faster observed dissociation rate in Tg-K104E vs. Tg-wild-type (WT) myosin and a smaller second-order ATP-binding rate for the K104E compared with WT myosin. Collectively, our data indicate that the mutation-induced changes in the interaction of myosin with actin during the contractionrelaxation cycle may contribute to altered contractility and the development of FHC. left ventricle; mutation of regulatory light chain; polarization of fluorescence INTERACTIONS OF MYOSIN WITH actin and associated proteins (sarcomeric proteins) are responsible for muscle contraction (19,24,48). These interactions include the mode of attachment of actin to myosin during different physiological states and the kinetics of conformational changes of myosin when interacting with actin. Familial hypertrophic cardiomyopathy (FHC) has been recognized to be caused by mutations in all major sarcomeric proteins of the heart, including the myosin regulatory light chain (RLC) (1). Compared with the myosin heavy chain, mutations in the RLC are rare, but often associated with malignant outcomes (18,27,41). It is therefore important to find out whether these FHC-linked mutations result in alterations of the mode of attachment of myosin to actin, and/or whether they lead to alterations of kinetics of acto-myosin interactions. These changes may ultimately contribute to the development of FHC. In this report we have focused on the K104E (lysine to glutamic acid) substitution in the myosin RLC expressed in the heart of transgenic (Tg) mice (25). The mutation was identified in a Danish population by Andersen et al. (2) and was associated with left ventricu...

show abstract

“…3). As the basic N-terminal region of A1-LC is apparently involved in the interaction with actin [35,361, an actinbinding property might be assumed for LC25. Results from experiments based on cosedimentation of LC25/28 with native actin filaments support this idea [15].…”

Section: Nkdeikqvwkeapieggkfdyvkfvrlikrgkeee(195)mentioning

confidence: 99%

Complete amino acid sequence of the regulatory light chain of obliquely striated muscle myosin from earthworm, Lumbricus terrestris

Serwe

Meyer

Craig

et al. 1993

European Journal of Biochemistry

View full text Add to dashboard Cite

Amino acid sequence analysis of the regulatory light chain of obliquely striated muscle myosin from earthworm, Lumbricus terrestris, was performed completely. The polypeptide consists of 195 amino acids with a calculated molecular mass of 21 943 Da. From the arrangement of amino acid residues, the first EF‐hand domain appears to be a specific Ca2+ ‐binding site. The unusually long N‐terminal region of about 40 amino acids, which is characterized by accumulation of basic amino acids, is similar to that of the myosin A1 catalytic light chain from rabbit skeletal muscle.

show abstract

Interaction of isozymes of myosin subfragment 1 with actin: effect of ionic strength and nucleotide

Cited by 67 publications

References 27 publications

Amplitude of the actomyosin power stroke depends strongly on the isoform of the myosin essential light chain

Amplitude of the actomyosin power stroke depends strongly on the isoform of the myosin essential light chain

Effect of a myosin regulatory light chain mutation K104E on actin-myosin interactions

Complete amino acid sequence of the regulatory light chain of obliquely striated muscle myosin from earthworm, Lumbricus terrestris

Contact Info

Product

Resources

About