Sugar transport by the bacterial phosphotransferase system. Nanosecond fluorescence studies of the phosphocarrier protein (HPr) labeled at the NH2-terminal methionine.
HPr is a low molecular weight, phosphocarrier protein of the Salmonella typhimurium phosphoenolpyruvate:glycose phosphotransferase system (PTS). This protein was alkylated with the fluorescent reagent (N-iodoacetylaminoethyl)-5-naphthylamino-1-sulfonate under conditions which favor alkylation of the thioether linkage in methionine residues (Link, T. P. and Stark, G. R. (1968) J. Biol. Chem. 243, 1082-1088) to give the corresponding sulfonium derivatives. The isolated fluorescent protein (95-100% pure) was as active as native HPr both as a phosphoryl acceptor protein (phosphoenolpyruvate and Enzyme I of the PTS), and as a phosphocarrier protein in the phosphorylation of methyl alpha-glucoside by the complete PTS. The fluorescent label was shown to be predominantly, possibly exclusively, at the NH2-terminal methionine residue. The decay of the fluorescence intensity could be described in terms of a biexponential function with the time constants tau 1 approximately 7 ns, tau 2 approximately 15 ns, and a ratio of alpha 2/alpha 1 approximately 3 for the pre-exponential factors. The decay of the fluorescence emission anisotropy was found to be consistent with some internal motion of the probe, in addition to the rotation of the protein conjugate as a whole.
D001426:Bacterial Proteins; D001692:Biological Transport; D050260:Carbohydrate Metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D007700:Kinetics; D008715:Methionine; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000378:metabolism*; D012486:Salmonella typhimurium / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014316:Tritium
D001426:Bacterial Proteins; D002352:Carrier Proteins; D014316:Tritium; D008715:Methionine; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C030182:phosphocarrier protein HPr