The molecular dynamics of actin measured by a spin probe attached to lysine.
Rabbit skeletal muscle G-actin was labeled with a spin probe, 3-(5-fluoro-2,4-dinitroanilino)proxyl. Tryptic digestion of the labeled actin followed by ultrafiltration and ion-exchange column chromatography indicated that the label was attached to residue Lys-61. This residue is found within a 9-kDa N-terminal segment that is easily degraded by proteolytic enzymes. The rate of reduction of the nitroxide bond by ascorbate was measured to determine the accessibility of the probe to small molecules in the solvent. These experiments showed that label bound to G-actin was relatively inaccessible to ascorbate, suggesting that it is buried within the protein structure. Polymerization further decreased the accessibility of the probe. Replacing bound Ca2+ with Mn2+ decreased the observed intensity of the electron paramagnetic resonance signal, indicating the spin label is about 2 nm distant from the metal binding site on the actin molecule. Labels attached to G-actin displayed an absorption spectrum characteristic of rotational motion with a correlation time (tau c) of 7 X 10(-9) s, which is faster than that for the whole molecule. Labels attached to F-actin had a value of tau c, measured using saturation transfer electron paramagnetic resonance, of 2 X 10(-5) s, which shows that the probe has a greater degree of mobility than the filament. The binding of heavy meromyosin or troponin-tropomyosin to labeled actin resulted in a further increase in the rotational correlation times, with the greatest decrease in mobility (tau c = 1 X 10(-4) s) observed when both were bound. Together the above results suggest that the 9-kDa segment of actin is mobile relative to the rest of the molecule and that this mobility can be influenced by the binding of heavymeromyosin or troponin-tropomyosin.
D000199:Actins / Q000378:metabolism*; D000818:Animals; D001205:Ascorbic Acid; D002852:Chromatography, Ion Exchange; D003497:Cyclic N-Oxides; D004578:Electron Spin Resonance Spectroscopy; D007700:Kinetics; D008239:Lysine; D046911:Macromolecular Substances; D008970:Molecular Weight; D009132:Muscles / Q000378:metabolism*; D015879:Myosin Subfragments / Q000378:metabolism; D010084:Oxidation-Reduction; D010446:Peptide Fragments; D011817:Rabbits; D013113:Spin Labels; D014335:Tropomyosin / Q000378:metabolism; D014336:Troponin / Q000378:metabolism; D014462:Ultrafiltration
D000199:Actins; D003497:Cyclic N-Oxides; D046911:Macromolecular Substances; D015879:Myosin Subfragments; D010446:Peptide Fragments; D013113:Spin Labels; D014335:Tropomyosin; D014336:Troponin; C051281:3-(5-fluoro-2,4-dinitroanilino)-1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidine; D008239:Lysine; D001205:Ascorbic Acid