Conformational studies of a two-module fragment of nebulin and implications for actin association.
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 alpha-helical content by computer analysis, CD spectra of ND8 indicated only a maximum of 10% alpha-helix and 25% beta-sheet in a variety of buffers; (b) The presence of increasing concentrations of trifluoroethanol (TFE) promoted the formation of alpha-helix and ND8 contained 50% alpha-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 alpha-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.
Chen
M J
MJ
Department of Chemistry and Biochemistry, University of Texas at Austin 78712.
Wang
K
K
D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D002942:Circular Dichroism; D005454:Fluorescence Polarization; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry* / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence
D000199:Actins; D009124:Muscle Proteins; D010446:Peptide Fragments; C035620:nebulin