Related Experiment Videos
Unfolding domains and tryptophan accessibility of a 59 kDa coiled-coil light meromyosin
1Boston Biomedical Research Institute, 20 Staniford Street, Boston, MA 02114, USA.
Abstract:
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.
Insights
Light meromyosin (LMM) unfolding was studied using two peptides, LMM 77 and LMM 59. The findings reveal that tryptophan residues in LMM 77 are in distinct unfolding domains, with part of the myosin thick filament core exposed to solvent.
Area of Science:
- Biochemistry
- Structural Biology
- Muscle Physiology
Background:
- Light meromyosin (LMM) is a C-terminal peptide of myosin rod, forming a coiled-coil structure.
- LMM contains tryptophan residues crucial for studying its unfolding properties.
- Previous studies suggested LMM 77's tryptophan pairs reside in the least stable unfolding domain.
Purpose of the Study:
- To compare the thermal and denaturant unfolding of LMM 77 and a truncated LMM 59.
- To determine the location of tryptophan residue pairs within LMM unfolding domains.
- To investigate the accessibility of LMM within myosin filaments.
Main Methods:
- Comparative analysis of thermal and denaturant unfolding properties of LMM 77 and LMM 59.
- Tryptophan fluorescence spectroscopy to monitor unfolding transitions.
- Disulfide crosslinking of LMM 59 to verify domain assignments.
- Solute-quenching studies and electron microscopy to assess LMM accessibility in filaments.
Main Results:
- LMM 59 exhibited two unfolding transitions similar to LMM 77.
- Only the second unfolding transition of LMM 59 affected tryptophan fluorescence, indicating distinct domains for LMM 77's Trp pairs.
- Solute-quenching and electron microscopy showed LMM 59's core region remained accessible within filaments.
Conclusions:
- The two tryptophan pairs in LMM 77 are located in different unfolding domains.
- A portion of the myosin thick filament core is exposed to the solvent.
- This structural insight is vital for understanding myosin filament assembly and function.