Unfolding/refolding studies of the myosin rod

Abstract

The effect of guanidine hydrochloride on the gel-filtration chromatography, viscosity, far ultraviolet circular dichroism and fluorescence emission intensity of the myosin rod was studied under equilibrium conditions. The normalized transition curves for each of these methods were comparable with a midpoint at a guanidine hydrochloride concentration of 1.75 -2 M. The curves were not, however, superposable, suggesting that the loss of helix content and the dissociation of the two chains of the myosin rod were not tightly linked. Furthermore, they were unexpectedly independent of the protein concentration over 0.05-20 pM. These phenomena are interpreted takmg into account the large size of the molecule. A step-wise process is proposed as a model for the unfolding of the myosin rod.Myosin is a major component of the contractile apparatus of vertebrate skeletal muscle. It is composed of two globular heads flexibly joined to a rod-like tail. This tail is generally referred to as the myosin rod and consists of two a-helical, heavy polypeptide chains wound round one another in a lefthanded coiled coil (McLachlan and Karn, 1982;Cohen and Parry, 1990). It is involved in myosin thick-filament formation and may also play a role in muscular contraction.The myosin rod and some of its subfragments (the myosin subfragment-2 and light meromyosin) can be purified after proteolytic cleavage of whole myosin. The structural stability and flexibility of the rod and of some rod regions have been questioned for many years. Harrington and Rodgers (1984; references therein) have suggested that a part of the myosin tail near the junction between the light meromyosin and the subfragment-2 (the hinge region) can easily melt at approximately physiological temperatures, possibly to a random-coil conformation. Such melting would be expected to give rise to a reduction in the overall length of the molecule, and this is consistent with electron-microscope observations of the effect of temperature on the length of the myosin tail (Walker and Trinick, 1986;Walzthony et al., 1986). This transition could therefore be an important force-generating event in muscle contraction and the subfragment-2 region as well as the myosin head would contribute to force production in actively contracting muscle (Harrington et al., 1990;Sugi et al., 1992).It has recently been proposed that the myosin rod contains six independent domains (Privalov, 1982 ;Lopez-Lacomba et al., 1989;Bertazzon and Tsong, 1990). The Cterminus of the heavy chain has been shown to be unfolded and mobile , and to play a crucial role in myosin assembly Kalbitzer et al. (1991) have indicated that the two chains are not in exact register but are slightly staggered. Thus, the variability in the structure of the rod may affect its properties.Protein denaturatiodrenaturation experiments may indicate how a multidomain dimeric protein, such as the myosin rod, folds and how its subunits and domains acquire stability [for a review, see Jaenicke (1987)l. We studied the effect of guanidine hydrochl...

Kinetics of Folding of the Myosin Rod

“…1) and similar at any protein concentration. Their amplitudes depended on the concentration of Gdn/HCl and the final signal was comparable to that observed in equilibrium experiments (Nozais and BCchet, 1993). Between 2 M and 3 M Gdn/HCl, the abrupt fall of the intensity of the fluorescence during the dead time of the apparatus was followed by a relatively slow decrease which was fitted to a single exponential (Fig.…”

“…Our previous equilibrium studies of the denaturation of the myosin rod in Gdn/HCl determined the conditions in which the protein is unfolded (Nozais and BCchet, 1993). The half-transition concentrations for various physico-chemical changes of the protein as a function of GddHC1 concentration are equal to 1.75-2 M. In the G d d HCl range of 3-5 M, the circular dichroism spectrum of the myosin rod shows very little secondary structure and increased random-coil content compared with the native protein.…”

“…Myosin rod was prepared from chymotryptic digests of myosin according to Margossian and Lowey (1 982), except that the aqueous buffers also contained 1 mM dithiothreito1 and 1 mM EDTA. Myosin rod was characterized as previously described (Nozais and BCchet, 1993). Its purity was assessed by polyacrylamide gel electrophoresis under native or denaturing conditions (Nozais and Btchet, 1993), and also by HPLC gel filtration on a TSK-Gel G4000SW column (0.7.5 cmX30 cm).…”

“…HPLC gel filtration of rod was carried out as previously reported (Nozais and BCchet, 1993). The column used was a TSKGel G4000SW column (0.75 cmX30 cm), equilibrated with potassium phosphate buffer containing one of a series of Gdn/HCl concentrations.…”

“…Protein denaturation/renaturation experiments in guanidine hydrochloride (GddHCl) suggested internal structural variability of the myosin rod (Nozais and BCchet, 1993). This last study was made in equilibrium conditions but the kinetics of association of isolated unfolded chains and those of dissociation of the whole molecule were not determined.…”