Unfolding domains and tryptophan accessibility of a 59 kDa coiled-coil light meromyosin.
Light meromyosin (LMM 77), the C-terminal proteolytic peptide from myosin rod, is a 900 A coiled-coil that contains two pairs of tryptophan residues in d-positions of the heptad repeat (abcdefg)n. Previous studies showed that LMM 77 unfolded in two transitions and suggested that both Trp pairs were located in the least stable unfolding domain. Here, the thermal and denaturant unfolding properties of LMM 59, a recombinant N-terminal truncated LMM, containing only one of the Trp pairs, was compared to LMM 77. LMM 59 unfolded in two transitions with similar midpoints to the two transitions of LMM 77. However, only the second transition of LMM 59 affected the Trp fluorescence, indicating that the two pairs of Trp residues in LMM 77 are in different unfolding domains. Disulfide-crosslinked LMM 59 verified this assignment. Solute-quenching studies showed that the accessibility of the Trp in LMM 59 decreased only by 56% on forming filaments. Electron micrographs indicated that all of LMM 59 is located within the core of a bipolar tactoid with the Trp-containing region the most accessible to negative strain, in agreement with the solute-quenching studies. This suggests that part of the core of the myosin thick filament is appreciably exposed to solvent.
Zhou
X
X
Boston Biomedical Research Institute, 20 Staniford Street, Boston, MA 02114, USA.
Maéda
Y
Y
Mabuchi
K
K
Lehrer
S S
SS
D002942:Circular Dichroism; D003432:Cross-Linking Reagents; D004220:Disulfides / Q000737:chemistry; D006358:Hot Temperature; D008854:Microscopy, Electron; D008970:Molecular Weight; D015879:Myosin Subfragments / Q000737:chemistry* / Q000648:ultrastructure; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000648:ultrastructure; D012997:Solvents; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry; D014508:Urea
D003432:Cross-Linking Reagents; D004220:Disulfides; D015879:Myosin Subfragments; D011994:Recombinant Proteins; D012997:Solvents; D014364:Tryptophan; D014508:Urea