Isolating and localizing ATP-sensitive tryptophan emission in skeletal myosin subfragment 1.
The fluorescence intensity difference between rabbit skeletal myosin subfragment 1 (S1) and nucleotide-bound or trapped S1 isolates ATP-sensitive tryptophans (ASTs) emission from the total tryptophan signal. Neutral (acrylamide) quenching of the ASTs is sensitive to the binding or trapping of nucleotide to the active site of S1. Anion (I(-)) quenching of the ASTs, sensitive to charge separation in the tryptophan micro environment, is negligible. These findings suggest the ASTs sense conformational change during ATPase from negatively charged surroundings. Specific chemical modifications of S1 identified the location of the ASTs. Trp131 was quenched by chemical modification, and its emission was isolated by taking the intensity difference between unmodified and modified S1. Trp131 fluorescence intensity and quenching constant do not distinguish among the bound or trapped nucleotides, suggesting that the vicinity of Trp131 does not change conformation during the ATPase cycle and eliminating Trp131 as an AST. Trp510 fluorescence was quenched by 5'-iodoacetamidofluorescein (5'IAF) modification of the reactive thiol (SH1) of S1. The tryptophan emission enhancement increment due to active site trapping decreases linearly with SH1 modification and extrapolates to 0 for 100% modification. These data identify Trp510 as the primary AST in skeletal S1 in agreement with observations from Dictyostelium (Batra and Manstein (1999) Biol. Chem. 380, 1017-1023) and smooth muscle S1 (Yengo et al. (2000) Biophys. J. 78, 242A). With Trp510 identified as the sole AST, fluorescence difference spectroscopy provides a novel means to monitor the concentration of myosin transient intermediates in ATP hydrolysis.
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Department of Biochemistry and Molecular Biology, Mayo Foundation, 200 First Street Southwest, Rochester, Minnesota 55905, USA.
Burghardt
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D020106:Acrylamide / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry*; D000818:Animals; D001665:Binding Sites; D008956:Models, Chemical; D018482:Muscle, Skeletal / Q000737:chemistry* / Q000378:metabolism; D015879:Myosin Subfragments / Q000737:chemistry* / Q000378:metabolism; D009218:Myosins / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011789:Quantum Theory; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013452:Sulfonium Compounds / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism
D015879:Myosin Subfragments; D013452:Sulfonium Compounds; D020106:Acrylamide; C024586:dimethyl(2-hydroxy-5-nitrobenzyl)sulfonium; D014364:Tryptophan; D000255:Adenosine Triphosphate; D009218:Myosins