Tryptophan 512 is sensitive to conformational changes in the rigid relay loop of smooth muscle myosin during the MgATPase cycle.
To examine the structural basis of the intrinsic fluorescence changes that occur during the MgATPase cycle of myosin, we generated three mutants of smooth muscle myosin motor domain essential light chain (MDE) containing a single conserved tryptophan residue located at Trp-441 (W441-MDE), Trp-512 (W512-MDE), or Trp-597 (W597-MDE). Although W441- and W597-MDE were insensitive to nucleotide binding, the fluorescence intensity of W512-MDE increased in the presence of MgADP-berellium fluoride (BeF(X)) (31%), MgADP-AlF(4)(-) (31%), MgATP (36%), and MgADP (30%) compared with the nucleotide-free environment (rigor), which was similar to the results of wild type-MDE. Thus, Trp-512 may be the sole ATP-sensitive tryptophan residue in myosin. In addition, acrylamide quenching indicated that Trp-512 was more protected from solvent in the presence of MgATP or MgADP-AlF(4)(-) than in the presence of MgADP-BeF(X), MgADP, or in rigor. Furthermore, the degree of energy transfer from Trp-512 to 2'(3')-O-(N-methylanthraniloyl)-labeled nucleotides was greater in the presence of MgADP-BeF(X), MgATP, or MgADP-AlF(4)(-) than MgADP. We conclude that the conformation of the rigid relay loop containing Trp-512 is altered upon MgATP hydrolysis and during the transition from weak to strong actin binding, establishing a communication pathway from the active site to the actin-binding and converter/lever arm regions of myosin during muscle contraction.
Yengo
C M
CM
Department of Molecular Physiology and Biophysics, College of Medicine, University of Vermont, Burlington 05405-0068, USA.
Chrin
L R
LR
Rovner
A S
AS
Berger
C L
CL
D000199:Actins / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D018360:Crystallography, X-Ray; D018076:DNA, Complementary / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009130:Muscle, Smooth / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D018995:Myosin Heavy Chains / Q000737:chemistry; D009218:Myosins / Q000737:chemistry*; D011487:Protein Conformation; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000378:metabolism*
D000199:Actins; D018076:DNA, Complementary; D020033:Protein Isoforms; D011994:Recombinant Proteins; D014364:Tryptophan; D017301:Ca(2+) Mg(2+)-ATPase; D018995:Myosin Heavy Chains; D009218:Myosins