Figure 2
(A) Superposition of the two 1HN,15N-HSQC spectra from the sample containing 1 mM 15N-labeled SH3 and 2 mM unlabeled Sos-1X′. The spectra were recorded during the time interval 8–32 min after initiation of the reaction (red) and 8 hrs later (green). The 3 pairs of peaks that have been used to determine the τ1/2 constant are labeled in the plot. (B) Chemical shift differences between (i) noncovalent SH3·Sos1-X′ complex and apo SH3 (Δnoncov, red bars) and (ii) covalent SH3:Sos1-X′ complex and SH3·Sos1-X′ (Δcov, green bars). Grey areas indicate the absence of data (proline residues, spectral overlaps). (C) Kinetic curves for residue Y37 reflecting the decrease in the intensity of the spectral peak associated with SH3·Sos1-X′ species (red points) and the concomitant increase in the intensity of the peak associated with SH3:Sos1-X′ species (green points). The time points in the graph correspond to the mid-points of the respective HSQC experiments. The best-fit τ1/2 value is indicated above the plot. (D) SDS-PAGE data reporting on the outcome of Sos1-X′ conjugation to SH3 domain. Reaction time 20 min, the pH conditions are marked underneath the lanes. (E) Sequence coverage based on LC-MS/MS experimental data and PEAKS Studio interpretation. Tryptic peptides with modifications are labelled.
