Thermal unfolding of myosin rod and light meromyosin: circular dichroism and tryptophan fluorescence studies

Abstract

Rabbit skeletal myosin rod, which is the coiled-coil alpha-helical portion of myosin, contains two tryptophan residues located in the light meromyosin (LMM) portion whose fluorescence contributes 27% to the fluorescence of the entire myosin molecule. The temperature dependence of several fluorescence parameters (quantum yield, spectral position, polarization) of the rod and its LMM portion was compared to the thermal unfolding of the helix measured with circular dichroism. Rod unfolds with three major helix unfolding transitions: at 43, 47, and 53 degrees C, with the 43 and 53 degrees C transitions mainly located in the LMM region and the 47 degrees C transition mainly located in the subfragment 2 region. The fluorescence study showed that the 43 degrees C transition does not involve the tryptophan-containing region and that the 47 degrees C transition produces an intermediate with different fluorescence properties from both the completely helical and fully unfolded states. That is, although the fluorescence of the 47 degrees C intermediate is markedly quenched, the tryptophyl residues do not become appreciably exposed to solvent until the 53 degrees C transition. It is suggested that although the intermediate that is formed in the 47 degrees C transition contains an extensive region which is devoid of alpha-helix, the unfolded region is not appreciably solvated or flexible. It appears to have the properties of a collapsed nonhelical state rather than a classical random coil.

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

“…King et al also indicated that the decrease in the intrinsic fluorescence intensity was due to a helical-helix of the rod [23]. The coiled-coil polypeptide structure of the rod loosens with heating and separates into single peptide chains and a red shift of the maximum wavelength of 10 nm has been observed [24]. The present study indicated that the red shift of the intrinsic fluorescence spectrum was only 4 nm under a pressure of 400 MPa ( Table 1).…”

Effect of ultrasonication on secondary structure and heat induced gelation of chicken myofibrils

“…3, the CD spectrum showed two minima at 210 and 222 nm, indicating the presence of a-helical structure (Greenfield 2007). As myosin constitutes major portion of myofibrillar proteins, the a-helix conformation is predominantly determined by supercoiled a-helix structure of myosin tail (King and Lehrer 1989;Kristinsson and Hultin 2003). Structurally, myosin is composed of two globular heads and a rod-like tail (Harington and Rodger 1984).…”

Distribution and evolution of stable single α-helices (SAH domains) in myosin motor proteins

“…The corresponding regions in the other mammalian muscle myosin homologs contain a conserved tryptophan at a “d” position (S6 Fig). This is one of the two conserved tryptophans in the muscle myosin rod region that were shown to be appreciably exposed to solvent and to be located in the least stable parts of the fibrous region [38–40]. Introducing a highly charged region in mammalian Mhc14 myosins could be an alternative solution to destabilize this part of the coiled-coil region.…”