On the Alkali Light Chains of Vertebrate Skeletal Myosin

Abstract

The interaction of alkali light chains of vertebrate skeletal myosin with nucleotides and KCl has been examined by chemical modifications of these proteins, by direct binding measurements, and in circular dichroism studies. The reactivities of the single thiol groups in the isolated alkali light chains A1 and A2 have been studied by reacting these proteins with 5,5′‐dithiobis(2‐nitrobenzoic acid) (Nbs2). MgATP and MgADP reduced the reactivities of thiol groups while high concentrations of KCl increased them. Subsequent equilibrium dialysis experiments verified the presence of a low‐affinity nucleotide binding site per each alkali subunit. Circular dichroism measurements revealed that KG induced local (around phenylalanines) and overall (α‐helical content) conformational changes of equal magnitude in the two alkali light chains. However, salt induced different conformational changes in the subfragment 1 isoenzymes, S‐1(A1) and S‐1(A2). This differential salt effect on the S‐1 isoenzymes was confirmed by comparing their thermal stability in different salt conditions. At low KCl concentrations (5 mM), S‐1(A1) was found to be considerably more heat labile than S‐1(A2); at higher salt levels (50 mM KCl) the stability of S‐1(A1) approached that of S‐1(A2). These experiments are discussed in terms of the relationship between the alkali subunits and the ATP and the actin‐binding sites of myosin.

Single molecule turnover of fluorescent ATP by myosin and actomyosin unveil elusive enzymatic mechanisms

“…The hypothesis of nonspecific binding, also of nonfluorescent ATP, outside the myosin active site is consistent with experimental results by others [61][62][63] which however, suggest fewer sites than our equilibrium dialysis experiments with Alexa-ATP. Nonspecific ATP binding to several sites outside the active site is also supported by results using the bioinformatics tool ATPint 64 (Fig.…”

Effects of Myosin "Essential" Light Chain A1 on the Aggregation Properties of the Myosin Head

“…Namely, it was shown that, at low ionic strength, S1(A1) aggregates at a substantially lower temperature than S1(A2) [18, 19]. It seems possible that, due to its semirigid extended structure, the A1 N–terminal segment can participate not only in intramolecular interactions, but also in intermolecular interactions with the motor domains of other S1 molecules.…”

“…It seems possible that, due to its semirigid extended structure, the A1 N–terminal segment can participate not only in intramolecular interactions, but also in intermolecular interactions with the motor domains of other S1 molecules. However, it should be noted that all previous experiments on S1 isoforms aggregation were carried out at very low ionic strengths and high protein concentrations [18, 19]. Unfortunately, nobody has undertaken a more thorough investigation of the thermal aggregation of S1 isoforms and the role of A1 N–terminal extension in this process.…”