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Fig. 1 | BMC Biology

Fig. 1

From: Molecular insights into substrate recognition and catalytic mechanism of the chaperone and FKBP peptidyl-prolyl isomerase SlyD

Fig. 1

Isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) binding studies. Typical ITC data are shown for binding of peptides to TtSlyD: a Binding of the S2 peptide to full-length TtSlyD (TtSlyDFL). b Binding of the S2-long2 peptide to TtSlyDFL. c Binding of the S2-short6 peptide to TtSlyDFL. d Binding of the S2-W23A peptide to a TtSlyD construct in which the insert-in-flap (IF) domain is replaced by the flap loop from human FKBP12 (TtSlyDΔIF). Upper panels: raw heat pulse data. Lower panels: Integrated heat changes upon binding plotted against the peptide/protein concentration ratio resulting in differential binding isotherms that can be adequately described by a two-site (panels a and b) or a one-site (panels c and d) binding model. Resulting binding parameters are summarized in Table 2. e NMR-titration of 15N-labeled TtSlyDFL with the S2-P25A peptide at 25 °C. The relative change in chemical shift is plotted versus the total concentration of added peptide, with red dots corresponding to residues in the IF domain (S77, A78, V85, and V86) and blue dots to the FKBP domain (G46, F128, and A138). The error bars indicate the standard error. The black solid curve represents the theoretical binding isotherm calculated using KD1 = 0.13 μM and the dashed curve represents the theoretical binding isotherm calculated using KD2 = 7.0 μM, as obtained from the ITC measurements (Table 2). f Mean weighted chemical shift changes are plotted against residue number. Resonances completely broadened by peptide binding are indicated by gray bars covering the full vertical scale. The cut-off of 0.1 ppm is shown as a red dotted line. g Residues with a shift difference >0.1 ppm or completely broadened are highlighted in red on the structure of the TtSlyDFL:S2 complex [PDB: 4ODL], with the peptide shown in blue

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