Trp-512 in smooth muscle myosin is sensitive to conformational changes during the MgATPase cycle, with fluorescence intensity increasing by 30-36% in the presence of nucleotides compared to rigor.
Trp-512 in the rigid relay loop of smooth muscle myosin is the sole ATP-sensitive tryptophan residue, altering conformation upon MgATP hydrolysis and establishing a communication pathway during muscle contraction.
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 et al. (2000) studied this question. Smooth muscle myosin motor domain essential light chain mutants vs. Wild type-MDE / rigor state was evaluated on Fluorescence intensity changes. Trp-512 in smooth muscle myosin is sensitive to conformational changes during the MgATPase cycle, with fluorescence intensity increasing by 30-36% in the presence of nucleotides compared to rigor.