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obs is the pseudo-first-order rate constant for cis-trans isomerization in the presence of PPlase, as described [G. Fischer, H. Bang, C. Mech, Biomed. Biochim. Acta 43, 1101 (1984); J. L. Kofron et al., Biochemistry 30, 6127 (1991)]. The affinity of Pin1 for peptides was measured as described [M. Schutkowski, S. Wöllner, G. Fischer, Biochemistry 34, 13016 (1995)].
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Molecular model-building was based on the coordinates of the Pin1 structure (9). The phosphate of pS in the modeled peptide was superimposed on the cocrystallizing sulfate ion in the original Pin1 structure, and Pro residue displacements were minimized with respect to the Ala-Pro ligand in the original Pin1 structure. Figures were made with the programs GRASP [A. Nicholls, K. Sharp, B. Honig, Proteins 11, 281 (1991)], Molscript, and Raster3d.
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Point mutations were introduced into Pin1 by polymerase chain reaction-based techniques and verified by DNA sequencing. The mutant proteins were expressed and purified as described (4, 7).
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26
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note
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m values for the phosphorylated versus unphosphorylated substrates is 19,400/7, which is about equal to the ratio of 1120/ <1 for the H59A mutant.
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Lysates of interphase and mitotic HeLa cells were incubated with GST or GST-Pin1 beads and the precipitated proteins subjected to immunoblot analysis with various antibodies, as described (7). S6 kinase antibodies are from N. Terada (National Jewish Medical and Research Center) and Upstate Biotechnology and Rab4 antibodies are from Santa Cruz Biotechnology.
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note
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We thank M. Berne, A. Bernhardt, and B. Hökelmann for peptide synthesis and sequencing and N. Terada for the antibodies to S6 kinase. M.B.Y. is a Howard Hughes Physician-Scientist Fellow. Supported by Deutsche Forschungsgemeinschaft, Fonds der Chemischen Industrie, and Boehringer Ingelheim Stiftung to G.F., and NIH grants GM56203 to L.C.C. and GM56230 to K.P.L.
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