Structural and kinetic studies of the 10 S<==>6 S transition in smooth muscle myosin.
The conformational transitions that smooth muscle myosin undergoes after nucleotide binding have been examined using fluorescently labeled nucleotides and regulatory light chain. The 10 S conformation of smooth muscle myosin could be induced by addition of 1-N6-ethenoadenosine or mant ADP plus beryllium fluoride, as well as by mant adenosine 5'-(beta,gamma-iminotriphosphate) (AMPPNP). Fluorescence lifetime studies using 1-N6-ethenoadenosine plus beryllium fluoride reveal two components for both (10 S)- and (6 S)-myosins, with little difference in the values of these lifetimes, their fractional amplitudes, or solute accessibilities. Anisotropy decay studies of myosin-mant nucleotide complexes demonstrate that the rotational correlation time for (10 S)-myosin is nearly 4-fold longer than that for (6 S)-myosin. Qualitatively similar results were obtained with a 5-[[[2(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid fluorescent probe attached to the regulatory light chain. Mant AMPPNP can be trapped in the active site by (10S)-myosin. Actin accelerates this release rate by 40-50-fold. These studies reveal: 1) reduction in nucleotide release rate by converting (6S) to (10S)-myosin is not due to a reduction in solute accessibility of the nucleotide 2) the heads in (10 S)-myosin are rigidly attached to the rest of the molecule, while in (6 S)-myosin, they have segmental flexibility, 3) regulatory light chain phosphorylation mimics the effect of high salt in enhancing segmental flexibility of the myosin heads, and 4) actin can induce the unfolding of (10 S)-myosin in the absence of regulatory light chain phosphorylation.
Rosenfeld
S S
SS
Department of Neurology, University of Alabama at Birmingham 35294.
Xing
J
J
Rener
B
B
Lebowitz
J
J
Kar
S
S
Cheung
H C
HC
D020106:Acrylamide; D000178:Acrylamides; D000199:Actins / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000378:metabolism; D000266:Adenylyl Imidodiphosphate / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001608:Beryllium; D002645:Chickens; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D005459:Fluorides; D005895:Gizzard, Avian; D007700:Kinetics; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000378:metabolism*; D009218:Myosins / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D009282:Naphthalenesulfonates; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D013050:Spectrometry, Fluorescence
D000178:Acrylamides; D000199:Actins; D017931:DNA Primers; D005456:Fluorescent Dyes; D009282:Naphthalenesulfonates; D011994:Recombinant Proteins; D020106:Acrylamide; D000266:Adenylyl Imidodiphosphate; C035982:beryllium fluoride; D000244:Adenosine Diphosphate; C006421:1,5-I-AEDANS; D009218:Myosins; D001608:Beryllium; D005459:Fluorides