Nucleotide dependent intrinsic fluorescence changes of W29 and W36 in smooth muscle myosin.
The intrinsic fluorescence of smooth muscle myosin is sensitive to both nucleotide binding and hydrolysis. We have examined this relationship by making MDE mutants containing a single tryptophan residue at each of the seven positions found in the wild-type molecule. Previously, we have demonstrated that a conserved tryptophan residue (W512) is a major contributor to nucleotide-dependent changes of intrinsic fluorescence in smooth muscle myosin. In this study, an MDE containing all the endogenous tryptophans except W512 (W512 KO-MDE) decreases in intrinsic fluorescence upon nucleotide binding, demonstrating that the intrinsic fluorescence enhancement of smooth muscle myosin is not solely due to W512. Candidates for the observed quench of intrinsic fluorescence in W512 KO-MDE include W29 and W36. Whereas the intrinsic fluorescence of W36-MDE is only slightly sensitive to nucleotide binding, that of W29-MDE is paradoxically both quenched and blue-shifted upon nucleotide binding. Steady-state and time-resolved experiments suggest that fluorescence intensity changes of W29 involve both excited-state and ground-state quenching mechanisms. These results have important implications for the role of the N-terminal domain (residues 1-76) in smooth muscle myosin in the molecular mechanism of muscle contraction.
van Duffelen
Marilyn
M
Department of Molecular Physiology and Biophysics, University of Vermont, College of Medicine, Burlington, Vermont 05405-0068, USA.
Chrin
Lynn R
LR
Berger
Christopher L
CL
D000199:Actins / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D018360:Crystallography, X-Ray; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006801:Humans; D006868:Hydrolysis; D007313:Insecta; D007700:Kinetics; D008958:Models, Molecular; D009119:Muscle Contraction; D009130:Muscle, Smooth / Q000378:metabolism*; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009711:Nucleotides / Q000737:chemistry; D011485:Protein Binding; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D024745:Smooth Muscle Myosins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods*; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry
D000199:Actins; D018076:DNA, Complementary; D009711:Nucleotides; D020033:Protein Isoforms; D011506:Proteins; D014364:Tryptophan; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D024745:Smooth Muscle Myosins