Thermal unfolding of myosin rod and light meromyosin: circular dichroism and tryptophan fluorescence studies.
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.
King
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Department of Muscle Research, Boston Biomedical Research Institute, Massachusetts 02114.
Lehrer
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D000818:Animals; D002942:Circular Dichroism; D015879:Myosin Subfragments; D009218:Myosins; D010446:Peptide Fragments; D011487:Protein Conformation; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013696:Temperature; D014364:Tryptophan
D015879:Myosin Subfragments; D010446:Peptide Fragments; D014364:Tryptophan; D009218:Myosins