Nebulin, a family of giant myofibrillar proteins of 700-900 kDa, has been proposed as a length-regulating template for the thin filaments of skeletal muscle. In the present study, the conformational states of a two-module nebulin fragment (ND8) were investigated by circular dichroism, steady-state fluorescence, fluorescence lifetime, and one-dimensional and two-dimensional NMR techniques. We observed the following: (a) Contrary to the predicted high α-helical content by computer analysis, CD spectra of ND8 indicated only a maximum of 10% α-helix and 25% β-sheet in a variety of buffers; (b) The presence of increasing concentrations of trifluoroethanol (TFE) promoted the formation of α-helix and ND8 contained 50% α-helix at 67% TFE; (c) Measurement of fluorescence lifetime, anisotropy, and CD of tyrosines in ND8 in various concentrations of TFE indicates that tyrosines are incorporated into the newly formed helical segments; (d) Preliminary correlation spectroscopy and nuclear Overhauser enhancement spectroscopy NMR spectra of ND8 confirmed the formation of α-helical structure and identified sites of helical segments in 30% TFE around the highly conserved tyrosine residues. These data suggest that the conformation of nebulin and its mode of interaction with actin are distinct from the helical tropomyosin.
ASJC Scopus subject areas
- Molecular Biology