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. 2009 Dec 12;285(8):5802–5814. doi: 10.1074/jbc.M109.068130

FIGURE 4.

FIGURE 4.

Effect of binding partners on EB1c chain exchange. A, shown is the time-dependent increase of the fluorescence signal at 527 nm (excitation at 434 m) after mixing equimolar amounts (10 μm each) of CFP-EB1c and YFP-EB1c at 37 °C in the absence of a binding partner and in the presence of either 591 μm ClipCG2, 65 μm p150CG, 23 μm p150CG(A49M), or 116 μm APCp1. The concentrations of the EB1c binding partners have been adjusted so that 95% of the EB1c is present in complexed form when taking the Kd values of the individual binding partners into account (15, 33). a.u., arbitrary units. B, shown are amide 1H exchange protection factors of EB1c in the absence (blue) and presence (red) of an equimolar amount of APCp1. No data are shown for residues 207, 211, and 215 in the complex because of resonance overlap and for the residues 237, 256, and 261 in both data sets because these residues are prolines. Residues 191–208 and 231–241 from the free EB1c and residues 191–202, 231–241, and 249–268 from EB1c in complex with APCp1 are not shown because their amide protons have completely exchanged before the first experiment could be started (about 10 min), corresponding to log p values ≤3. The locations of the helices α1 and α2 in the structure of the EB1c homodimer (PDB entry 1WU9) are indicated. C, shown is the location of slowly exchanging amide hydrogens in the structure of the free EB1c dimer (left) and the EB1c dimer in complex with a peptide derived from the microtubule-actin cross-linking factor (right), which was shown to form a closely similar complex to the one with APCp1 (14). The backbone is shown in gray, and selected residue positions are indicated. The exchange data are represented by color-coded spheres at the position of the backbone N atoms: light blue, fast exchanging 1H, not observed in the first exchange spectrum; yellow, log p < 4; orange, log p = 4.0–5.0; red, log p > 5. The locations of the bound peptides are indicated by a green tube representing a spline function through the Cα atom coordinates.