Smooth muscle myosin mutants containing a single tryptophan reveal molecular interactions at the actin-binding interface.
Elucidation of the molecular details of the cyclic actomyosin interaction requires the ability to examine structural changes at specific sites in the actin-binding interface of myosin. To study these changes dynamically, we have expressed two mutants of a truncated fragment of chicken gizzard smooth muscle myosin, which includes the motor domain and essential light chain (MDE). These mutants were engineered to contain a single tryptophan at (Trp-546) or near (Trp-625) the putative actin-binding interface. Both 546- and 625-MDE exhibited actin-activated ATPase and actin-binding activities similar to wild-type MDE. Fluorescence emission spectra and acrylamide quenching of 546- and 625-MDE suggest that Trp-546 is nearly fully exposed to solvent and Trp-625 is less than 50% exposed in the presence and absence of ATP, in good agreement with the available crystal structure data. The spectrum of 625-MDE bound to actin was quite similar to the unbound spectrum indicating that, although Trp-625 is located near the 50/20-kDa loop and the 50-kDa cleft of myosin, its conformation does not change upon actin binding. However, a 10-nm blue shift in the peak emission wavelength of 546-MDE observed in the presence of actin indicates that Trp-546, located in the A-site of the lower 50-kDa subdomain of myosin, exists in a more buried environment and may directly interact with actin in the rigor acto-S1 complex. This change in the spectrum of Trp-546 constitutes direct evidence for a specific molecular interaction between residues in the A-site of myosin and actin.
Yengo
C M
CM
Department of Molecular Physiology and Biophysics, College of Medicine, University of Vermont, Burlington, VT 05405, USA.
Fagnant
P M
PM
Chrin
L
L
Rovner
A S
AS
Berger
C L
CL
D000199:Actins / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites; D002645:Chickens; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005895:Gizzard, Avian; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000378:metabolism; D009130:Muscle, Smooth / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D018994:Myosin Light Chains / Q000737:chemistry / Q000378:metabolism; D009218:Myosins / Q000737:chemistry* / Q000378:metabolism*; D010649:Phenylalanine; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017384:Sequence Deletion; D013050:Spectrometry, Fluorescence; D014364:Tryptophan
D000199:Actins; D018076:DNA, Complementary; D018994:Myosin Light Chains; D010649:Phenylalanine; D014364:Tryptophan; D009218:Myosins