Inactivation, subunit dissociation, aggregation, and unfolding of myosin subfragment 1 during guanidine denaturation

Abstract

The effect of guanidine hydrochloride on ATPase activity, gel filtration, turbidity, exposure of thiol groups, far-UV circular dichroism, and the fluorescence emission intensity of myosin subfragment 1 (S-1) was studied under equilibrium conditions. It was found that the denaturation process involves several intermediate states. The enzymatic activity of S-1 is at first lost at very low concentrations of GdnHCl (lower than 0.5 M). At a slightly higher GdnHCl concentration (about 0.5 M), the light chains dissociate and this dissociation is closely followed by the formation of aggregates between the naked heavy chains of S-1 molecules in the guanidine hydrochloride range of concentrations 0.5-1 M. At GdnHCl concentrations above 1 M, aggregates gradually disappear and S-1 loses its secondary and tertiary structures. These phenomena are partly reversible, and ATPase activity is only partially recovered under highly limited conditions. These results are discussed in relation to the nature of myosin subunit assembly. The head fragment of 20 kDa is thus suggested to be implicated in the binding of light chain to heavy chain and in the self-association of free heavy chains.

Unfolding/refolding studies of the myosin rod

“…The effect of guanidine hydrochloride on the buffer pH was assessed as indicated in the study of the unfolding of the myosin head (Nozais et al, 1992). The incubation was followed by fluorescence studies, gelfiltration chromatography or other measurements as indicated below; these assays were carried out with the undiluted sample at 20°C as rapidly as possible.…”

αB-Crystallin Maintains Skeletal Muscle Myosin Enzymatic Activity and Prevents its Aggregation under Heat-shock Stress

“…Thermally and chemically induced unfolding of myosin in vitro appears irreversible, and it has been proposed that chaperones are required for proper folding of the motor. [19][20][21] In vivo, chaperones HSC-70 and HSP-90 are associated specifically with early folding stages of myosin in striated muscles. 21 Biochemical studies on isolated contractile proteins are essential for understanding how aB-crystallin and other sHSPs maintain the structure and function of individual contractile elements.…”

Changes in rabbit skeletal myosin and its subfragments under high hydrostatic pressure

“…The red shift observed under a pressure of 400 MPa was only 4 nm ( Table 1), suggesting that the structural changes of myosin and S1 by pressurization differ from those induced by denaturant such as GuHCl. We observed that myosin and S1 were completely unfolded [21]. The center of spectral mass of the rod linearly decreased with elevating pressure to 400 MPa, and the subsequent pressure decrease from 400 to 0.1 MPa increased the center of spectral mass.…”