title: On the tryptophan residue of smooth muscle myosin that responds to binding of nucleotide.
abstract: Initially, we asked which (of 10) smooth muscle myosin head residues responds to MgADP or MgATP binding with enhanced fluorescence emission (Trp-441 and Trp-512 were leading candidates)? To decide, we prepared sham-mutated smooth muscle heavy meromyosin (HMM), W441F HMM, and W512F HMM. On adding MgATP, emission of wild-type and W441F HMMs increased by 25-27%, but that of W512F HMM by 5%. So, in myosin, 512 is the "sensitive Trp." Unexpectedly, properties of W512F HMM [elevated Ca(2+)-ATPase, depressed EDTA (K(+))-ATPase, no regulation of its basal or actin-activated Mg(2+)-ATPase by phosphorylation of its "regulatory" light chain, limited actin activation, and inability to move actin filaments in a motility assay] are strikingly like those of smooth muscle myosin reacted at Cys-717 with thiol reagent. From crystallography-based [Houdusse, A., Kalabakis, V. N., Himmel, D., Szent-Györgyi, A. G. & Cohen, C. (1999) Cell 97, 459-470] simulations, we found that in wild-type HMM with MgADP added, Trp-512 is in a "hydrophobic pocket," but that pocket becomes distorted in W512F HMM. We think that there is a "path of influence" from 512 to 717 to the active site. We suggest that the mutational changes at 512 are transmitted along this path to Cys-717, where they induce changes similar to those caused by reacting wild-type HMM with thiol reagent.
authors.affiliation: Department of Structural Analysis, National Cardiovascular Center Research Institute, Fujishiro-dai, Suita, Osaka 565-8565, Japan. honishi@ri.ncvc.go.jp
authors.forename: H
authors.initials: H
authors.lastname: Onishi
authors.forename: K
authors.initials: K
authors.lastname: Konishi
authors.forename: K
authors.initials: K
authors.lastname: Fujiwara
authors.forename: K
authors.initials: K
authors.lastname: Hayakawa
authors.forename: M
authors.initials: M
authors.lastname: Tanokura
authors.forename: H M
authors.initials: HM
authors.lastname: Martinez
authors.forename: M F
authors.initials: MF
authors.lastname: Morales
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers; D009130:Muscle, Smooth / Q000378:metabolism*; D016296:Mutagenesis; D009218:Myosins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate; D009218:Myosins