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1.
Figure 7

Figure 7. Phosphotransfer from P-EINtr to NPr. From: DEUTERATION OF ESCHERICHIA COLI ENZYME INtr ALTERS ITS STABILITY.

Kinetics of the phosphotransfer were carried out as described in Methods. Filled circles, D- EINtr; filled squares, H-EINtr.

Grzegorz Piszczek, et al. Arch Biochem Biophys. ;507(2):332-342.
2.
Figure 5

Figure 5. Near UV circular dichroism of EINtr. From: DEUTERATION OF ESCHERICHIA COLI ENZYME INtr ALTERS ITS STABILITY.

The H and D forms of EINtr at 0.6 mg/ml were scanned (16 accumulations) in a Jasco J-715 spectrometer. In panel B, PEP (150 μM) and MgCl2 (2 mM) were added.

Grzegorz Piszczek, et al. Arch Biochem Biophys. ;507(2):332-342.
3.
Figure 11

Figure 11. Titration Calorimetry of EINtr(H356A) with NPr. From: DEUTERATION OF ESCHERICHIA COLI ENZYME INtr ALTERS ITS STABILITY.

Isothermal titration calorimetry was carried out as described in Materials and Methods. Panel A: The concentration of H-EINtr was 14 μM and that of NPr in the injection syringe was 380 μM. Panel B: The concentration of D-EINtr was 16.4 μM and that of NPr in the injection syringe was 380 μM.

Grzegorz Piszczek, et al. Arch Biochem Biophys. ;507(2):332-342.
4.
Fig. 1

Fig. 1. Sequence of EINtr(H356A). From: DEUTERATION OF ESCHERICHIA COLI ENZYME INtr ALTERS ITS STABILITY.

The sequence shown corresponds to the truncated form of EINtr preceded by a His-tag sequence and the aminoterminal Met deleted as described in Materials and Methods. Highlighted residues: His-tag sequence=orange; arginine residues=magenta; tryptophan residues=bright green; active site His356 changed to Ala=baby blue.

Grzegorz Piszczek, et al. Arch Biochem Biophys. ;507(2):332-342.
5.

Figure 10. Fluorescence effects on PEP and NPr binding to Trp mutants of EINtr. From: DEUTERATION OF ESCHERICHIA COLI ENZYME INtr ALTERS ITS STABILITY.

(A) PEP titration: Incubation conditions were as described for Fig. 13, except that the final concentrations of all the EINtr proteins used was 82 μg/ml. The baseline fluorescence intensities were: wild-type (filled squares), 42622; W186F (inverted triangles), 41324; W319F (triangles), 37487; W522F (circles), 28078. (B) NPr titration: incubation conditions were as in (A). The baseline fluorescence intensities were: wild-type, 42622; W186F, 72761; W319F, 87741; W522F, 51338.

Grzegorz Piszczek, et al. Arch Biochem Biophys. ;507(2):332-342.
6.
Figure 6

Figure 6. Autophosphorylation of EINtr. From: DEUTERATION OF ESCHERICHIA COLI ENZYME INtr ALTERS ITS STABILITY.

Preliminary autophosphorylation experiments (with 5 and 10 min incubation periods) were carried out to determine the maximum autophosphorylation of EINtr stocks of the dephosphorylated (see Materials and Methods) H and D forms. Kinetic studies were carried out as described in Methods. Filled circles, H-EINtr; filled squares, D-EINtr. The inset to the figure displays the same data with the time scale plotted logarithmically to clarify the kinetics.

Grzegorz Piszczek, et al. Arch Biochem Biophys. ;507(2):332-342.
7.
Figure 9

Figure 9. Iodide quench of the fluorescence of EINtr. From: DEUTERATION OF ESCHERICHIA COLI ENZYME INtr ALTERS ITS STABILITY.

Incubation mixtures (150 μl total volume) contained Tris·Cl, pH 8, 20 mM; KI and KCl, the sum of which was always 1 M; and EINtr(H356A) in the H form (0.98 μM) or D form (1.36 μM). Fluorescence was measured with excitation at 295 nm and emission at 340 nm. Fluorescence quenching was analyzed according to the Stern-Vollmer relationship (F0/F1) [35] where F0 is the fluorescence in the absence of quencher and F1 is the fluorescence in the presence of the indicated quencher concentration. Filled circles, D-EINtr; filled squares, H-EINtr. Upper panel, absence of PEP; lower panel, presence of 2 mM PEP and 2 mM MgCl2.

Grzegorz Piszczek, et al. Arch Biochem Biophys. ;507(2):332-342.
8.
Figure 4

Figure 4. Oligomerization state of EINtr. From: DEUTERATION OF ESCHERICHIA COLI ENZYME INtr ALTERS ITS STABILITY.

Light scattering analysis of H-EINtr was carried out as described in Materials and Methods. The gel filtration column was equilibrated with 20 mM Tris pH 8, 2 mM MgCl2, 150 mM NaCl. 100 μl of protein at 0.5 mg/ml was injected. When the protein was run in the presence of PEP, the column was pre-equilibrated with the buffer described above supplemented with 150 μM PEP. An aliquot (100 μl of protein at 0.5 mg/ml in the PEP supplemented buffer) was injected onto the column. The masses of the different regions (denoted by the red line) of the eluate were determined. Inset: Light scattering analysis of D-EINtr. A 100 μl sample of protein (0.5 mg/ml) was injected onto the column equilibrated with the Tris, MgCl2, NaCl buffer described above. The horizontal scale is time and the vertical scale is light scattering intensity.

Grzegorz Piszczek, et al. Arch Biochem Biophys. ;507(2):332-342.
9.
Figure 3

Figure 3. Thermal unfolding of EINtr. From: DEUTERATION OF ESCHERICHIA COLI ENZYME INtr ALTERS ITS STABILITY.

Panel A: Thermally induced changes in fluorescence. EINtr(H356A) in the H and D forms (25 μg/ml) in 20 mM Tris·Cl (pH 8), 2 mM MgCl2, 5 mM NaCl, 0.1 mM β-mercaptoethanol, 0.05 mM EDTA and 0.5% glycerol in a total volume of 1 ml were scanned through the indicated temperature range at a scan rate of 30 °C/h. Where indicated (H–P and D–P), the samples also contained 150 μM PEP. Excitation was at 295 nm and emission at 340 nm. Panel B: DSC scans (see Materials and Methods) of EINtr(H356A) H and D forms. The proteins were dialyzed against 10 mM NaPO4, pH 8 and adjusted to a concentration of 300 μg/ml.

Grzegorz Piszczek, et al. Arch Biochem Biophys. ;507(2):332-342.
10.

Figure 8. Effect of PEP and NPr concentration on EINtr fluorescence. From: DEUTERATION OF ESCHERICHIA COLI ENZYME INtr ALTERS ITS STABILITY.

(A) PEP titration: Reaction mixtures (1 ml volume) contained: Tris·Cl, pH 8, 20 mM; MgCl2, 2 mM, EINtr(H356A), 0.5 μM. The baseline fluorescence intensities were: H, 4960; D, 5150. Fluorescence at 340 nm was measured at 37 °C after the indicated additions of PEP. The increase in fluorescence is plotted against the PEP concentration. An incubation mixture without added EINtr showed little fluorescence and no change in fluorescence after addition of PEP (data not shown). H form, squares; D form, circles. The measured fluorescence intensities were expressed as the % increase in fluorescence over the baseline. (B) NPr titration: Reaction mixtures (1 ml volume) contained: Tris·Cl, pH 8, 20 mM; MgCl2, 2 mM, EINtr(H356A), 0.5 μM. The baseline fluorescence intensities were: H, 5985; D, 4986. Fluorescence at 340 nm was measured at 37 °C after the indicated additions of NPr. The increase in fluorescence is plotted against the NPr concentration. An incubation mixture without added EINtr showed some fluorescence change after addition of NPr (data not shown). NPr has no Trp or Tyr residues but does have 3 Phe residues. The sequence is: MTVKQTVEITNKLGMHARPAMKLFELMQGFDAEVLLRNDEGTEAEANSVIALLMLDSA KGRQIEVEATGPQEEEALAAVIALFNS. The data were corrected for this change due to the NPr fluorescence. H-EINtr, filled squares; D-EINtr, open circles. The measured fluorescence intensities were expressed as the % increase in fluorescence over the baseline. (C) Effect of H-EINtr concentration on the PEP-dependent fluorescence change. Experimental conditions were as described above, except that PEP, where added, was at 2 mM. The EINtr(H356A) concentration was varied, as indicated.

Grzegorz Piszczek, et al. Arch Biochem Biophys. ;507(2):332-342.
11.
Fig. 2

Fig. 2. Limited proteolysis of EINtr. From: DEUTERATION OF ESCHERICHIA COLI ENZYME INtr ALTERS ITS STABILITY.

Approximately 65 μg of either H-EINtr(H356A) or D-EINtr(H356A) was incubated with 0.1 μg trypsin in 10 mM Tris·Cl (pH 8), 100 mM NaCl, 2 mM β-mercaptoethanol, 1 mM EDTA and 10 % glycerol in a total volume of 75 μl at room temperature. A companion incubation mixture was supplemented with 56 μg of NPr 1–85. At the indicated times (0 to 90 mins, shown above the lanes), 7.5 μl aliquots were removed and mixed with 7.5 μl of 2× Laemmli sample buffer. The samples were run on 4–20% SDS-PAGE gradient gels (Invitrogen), then stained with Coomassie Blue R250 (panels A & B). Lanes labeled H correspond to H-EINtr and those labeled D correspond to D-EINtr. The stained gels were scanned in an Odyssey near infrared fluorescence scanner (Li-Cor Biosciences) and then the fluorescence of Coomassie Blue [41] was quantitated by the Odyssey software. The intensity of the bands (panels C through H) were measured after subtraction of the background of the gels. Panels C & D, patterns of the degradation of the intact protein, designated by the topmost arrowhead on the left side of panel A; panels E & F, relative amounts of the ~40 kDa peptide (identified as including residues 170–523, mass of 39,794.4, see fig. 2), designated by the center arrowhead; panels G & H, relative amounts of the ~25 kDa peptide (G524-L748, see fig. 2), designated by the lowest arrowhead. Solid lines with squares = H-EINtr; dashed lines with circles = D-EINtr.Panels A, C, E, and G correspond to proteolysis in the absence of NPr; panels B, D, F and H correspond to proteolysis in the presence of NPr.

Grzegorz Piszczek, et al. Arch Biochem Biophys. ;507(2):332-342.

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