Detection of ADAP-SH2 interactions by pull-down.

<p>(A) Pull-down experiment showing the interaction of ADAP and Rasa1b as an example. In this experiment, the SH2 protein Rasa1b was radioactively labeled by adding ¹⁴C-Leu to the synthesis reaction to allow visualization. Left panels: Detection of fluorescent ADAP in the SDS gel (excitation 633 nm) after electrophoresis, middle panels: Coomassie stain of SDS gel, right panels: Autoradiography of SDS gel with detection of radioactively labeled SH2 protein. ADAP-P: phosphorylated ADAP, ADAP-OH: non-phosphorylated ADAP, F: Flow-through on beads containing unbound ADAP and <i>E. coli</i> lysate proteins, w1, w2: wash fractions, E: strip fraction containing liberated complex of ADAP, SH2 and streptavidin. Bound and liberated ADAP is marked by an arrow, Rasa1b is marked by an asterisk, St: streptavidin. (B) results of pull-down screening. gray columns: ADAP-P, white columns: ADAP-OH. Error bars represent triplicates.</p

Supernatant depletion assay.

<p>(A) Titration of ADAP-P versus selected SH2 domains immobilized on streptavidin magnetic beads. (B) K_d values as they were estimated based on the plot shown in A. (C) In-gel fluorescence-detection showing complex depletion, decreasing SH2 concentration from left to right according to increasing numbers. For further explanation please refer to methods. ♦ RasaN, □ Fyn, ∆ SLP-76.</p

His-Tag purification of phosphorylated ADAP (ADAP-P) and non-phosphorylated ADAP (ADAP-OH).

<p>(A) Detection of fluorescent ADAP in SDS gel (excitation 633 nm), (B) Coomassie stain, (C) Western Blot with anti phosphotyrosine antibody. P: cell-free synthesis of ADAP after <i>in </i><i>vitro</i> phosphorylation, C: non-phosphorylated ADAP synthesis, F: flow-through, w1 and w2: wash fractions, E: purified ADAP.</p

Overview of the applied work-flow for the characterization of SH2-ADAP interactions.

<p>i) Amplification of Linear Templates encoding different SH2 proteins by two steps of PCR. ii) Synthesis of site-specifically biotinylated proteins. Subsequent immobilization on streptavidin-coated magnetic beads and pull-down with ADAP for validation and filtering of direct interactions. iii) Cloning of selected Linear Templates into the expression vector pIX3.0. iv) High yield synthesis of SH2 proteins without biotin and subsequent K_d value determination using Microscale Thermophoresis.</p

Quantitative interaction analysis.

<p>(A) Determined K_d values of the interaction of phosphorylated ADAP and YEEI with SH2. Deviations represent duplicates of the measurements. (B) Exemplary MST result for the interaction of phosphorylated YEEI (♦) and non-phosphorylated YEEI (◊) against RasaN. Error bars represent standard deviations of two measurements.</p

Completion of Proteomic Data Sets by Kd Measurement Using Cell-Free Synthesis of Site-Specifically Labeled Proteins

The characterization of phosphotyrosine mediated protein-protein interactions is vital for the interpretation of downstream pathways of transmembrane signaling processes. Currently however, there is a gap between the initial identification and characterization of cellular binding events by proteomic methods and the in vitro generation of quantitative binding information in the form of equilibrium rate constants (Kd values). In this work we present a systematic, accelerated and simplified approach to fill this gap: using cell-free protein synthesis with site-specific labeling for pull-down and microscale thermophoresis (MST) we were able to validate interactions and to establish a binding hierarchy based on Kd values as a completion of existing proteomic data sets. As a model system we analyzed SH2-mediated interactions of the human T-cell phosphoprotein ADAP. Putative SH2 domain-containing binding partners were synthesized from a cDNA library using Expression-PCR with site-specific biotinylation in order to analyze their interaction with fluorescently labeled and in vitro phosphorylated ADAP by pull-down. On the basis of the pull-down results, selected SH2’s were subjected to MST to determine Kd values. In particular, we could identify an unexpectedly strong binding of ADAP to the previously found binding partner Rasa1 of about 100 nM, while no evidence of interaction was found for the also predicted SH2D1A. Moreover, Kd values between ADAP and its known binding partners SLP-76 and Fyn were determined. Next to expanding data on ADAP suggesting promising candidates for further analysis in vivo, this work marks the first Kd values for phosphotyrosine/SH2 interactions on a phosphoprotein level