Unfolding domains in smooth muscle myosin rod.
Gizzard smooth muscle myosin rod, an alpha-helical coiled coil, exhibits two cooperative thermal or denaturant-induced helix unfolding transitions in solutions containing 0.6 M NaCl at neutral pH, when monitored by circular dichroism at 222 nm. The first smaller transition unfolds part of the subfragment 2 (S2) domain, and the main transition unfolds the remaining helix including the light meromyosin (LMM) domain. These unfolding domains were identified by monitoring the fluorescence of acrylodan, an environmentally sensitive fluorescence probe, and the ESR signal of a maleimide spin-label, sensitive to motion, both specifically attached to Cys 43 in the S2 region of the rod sequence. The identities of the domains were verified by studying the unfolding of the S2 and LMM coiled-coil peptides obtained by proteolytic cleavage of spin-labeled and unlabeled rod. The fluorescence of acrylodan-labeled rod indicated that although the S2 intermediate is unfolded, it is not in a random-coil conformation. The unfolded S2 region stabilized the LMM domain against unfolding, possibly by a direct interaction with the LMM region. Such an interaction may be involved in the salt- and phosphorylation-dependent 6S to 10S shift in configuration of the myosin molecule.
King
L
L
Department of Biochemistry, Chang Gung Medical College, Tao-Yuan, Taiwan, Republic of China.
Seidel
J C
JC
Lehrer
S S
SS
D015081:2-Naphthylamine / Q000031:analogs & derivatives; D000818:Animals; D002645:Chickens; D002942:Circular Dichroism; D004578:Electron Spin Resonance Spectroscopy; D005456:Fluorescent Dyes; D005895:Gizzard, Avian / Q000737:chemistry; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009130:Muscle, Smooth / Q000737:chemistry*; D009218:Myosins / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D006146:Guanidines; C038518:acrylodan; D015081:2-Naphthylamine; D009218:Myosins; D019791:Guanidine