A new method to specifically label thiophosphorylatable proteins with extrinsic probes. Labeling of serine-19 of the regulatory light chain of smooth muscle myosin.
We present a new method to specifically and stably label proteins by attaching extrinsic probes to amino acids that are thiophosphorylated by protein kinases and ATP gamma S. The method was demonstrated for labeling of a thiophosphorylatable serine of the isolated regulatory light chain of smooth muscle myosin. We stoichiometrically blocked the single thiol (Cys-108) either by forming a reversible intermolecular disulfide bond or by reacting with iodoacetic acid. The protein was stoichiometrically thiophosphorylated at Ser-19 by myosin light chain kinase and ATP gamma S. The nucleophilic sulfur of the protein phosphorothioate was coupled at pH 7.9 and 25 degrees C to the fluorescent haloacetate [3H]-5-[[2-[(iodoacetyl)-amino]ethyl]amino]naphthalene-1- sulfonic acid ([3H]IAEDANS) by displacement of the iodide. Typical labeling efficiencies were 70-100%. The labeling was specific for the thiophosphorylated Ser-19, as determined from the sequences of two labeled peptides isolated from a tryptic digest of the labeled protein. [3H]IAEDANS attached to the thiophosphorylated Ser-19 was stable at pH 3-10 at 25 degrees C, and to boiling in high concentrations of reductant. The labeled light chains were efficiently exchanged for unlabeled regulatory light chains of the whole myosin molecule. The resulting labeled myosin had normal ATPase activities in the absence of actin, indicating that the modification of Ser-19 and the exchange of the labeled light chain into myosin did not significantly disrupt the protein. The labeled myosin partially retained the elevated actin-activated Mg(2+)-ATPase activity which is characteristic of thiophosphorylated myosin. This indicates that labeling of the thiophosphate group with [3H]IAEDANS did not completely disrupt the functional properties of the thiophosphorylated protein in the presence of actin.
Facemyer
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Washington State University, Department of Biochemistry and Biophysics, Pullman 99164-4660.
Cremo
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D000818:Animals; D002645:Chickens; D007553:Isotope Labeling / Q000379:methods; D009130:Muscle, Smooth / Q000737:chemistry*; D009218:Myosins / Q000032:analysis*; D009282:Naphthalenesulfonates / Q000737:chemistry; D010455:Peptides / Q000302:isolation & purification; D010710:Phosphates / Q000737:chemistry*; D010766:Phosphorylation; D012680:Sensitivity and Specificity; D012694:Serine / Q000737:chemistry*; D013997:Time Factors; D014316:Tritium
D009282:Naphthalenesulfonates; D010455:Peptides; D010710:Phosphates; D014316:Tritium; D012694:Serine; C006421:1,5-I-AEDANS; D009218:Myosins; C035638:thiophosphoric acid