Iris Claußnitzer - Academia.edu (original) (raw)
Uploads
Papers by Iris Claußnitzer
<p>(A) Pull-down experiment showing the interaction of ADAP and Rasa1b as an example. In th... more <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 <sup>14</sup>C-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
<p>i) Amplification of Linear Templates encoding different SH2 proteins by two steps of PCR... more <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<sub>d</sub> value determination using Microscale Thermophoresis.</p
<p>(A) Detection of fluorescent ADAP in SDS gel (excitation 633 nm), (B) Coomassie stain, (... more <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
<p>(A) Titration of ADAP-P versus selected SH2 domains immobilized on streptavidin magnetic... more <p>(A) Titration of ADAP-P versus selected SH2 domains immobilized on streptavidin magnetic beads. (B) K<sub>d</sub> 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
<p>(A) Determined K<sub>d</sub> values of the interaction of phosphorylated ADA... more <p>(A) Determined K<sub>d</sub> 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
PLoS ONE, 2013
The characterization of phosphotyrosine mediated protein-protein interactions is vital for the in... more 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 (K d 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 K d 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 K d 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, K d 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 K d values for phosphotyrosine/SH2 interactions on a phosphoprotein level.
Organic & Biomolecular Chemistry, 2012
The bioorthogonal and chemoselective fluorescence labelling of several cell-free synthesized prot... more The bioorthogonal and chemoselective fluorescence labelling of several cell-free synthesized proteins containing a site-specifically incorporated azido amino acid was possible using different alkyne-functionalized Ru(II) bathophenanthroline complexes. We were able to achieve a selective labelling even in complex mixtures of proteins despite the fact that ruthenium dyes normally show a high tendency for unspecific interactions with proteins and are commonly used for total staining of proteins. Since the employed Ru complexes are extremely robust, photo-stable and highly sensitive, the approach should be applicable to the production of labelled proteins for single molecule spectroscopy and fluorescence-based interaction studies.
Angewandte Chemie International Edition, 2009
Analytical Biochemistry, 2014
Angewandte Chemie, 2009
Wir bedanken uns für finanzielle Unterstützung bei der Deutschen Forschungsgemeinschaft (DFG) im ... more Wir bedanken uns für finanzielle Unterstützung bei der Deutschen Forschungsgemeinschaft (DFG) im Rahmen des Emmy-Noether-Programms (HA 4468/2-1) und des SFB 765 sowie beim Fonds der Chemischen Industrie (FCI). Wir danken Dr. Dirk Schwarzer, Dr. mann und Wiebke Ahlbrecht für experimentelle Beiträge und sehr hilfreiche Diskussionen. Hintergrundinformationen zu diesem Beitrag sind im WWW unter http://dx.
<p>(A) Pull-down experiment showing the interaction of ADAP and Rasa1b as an example. In th... more <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 <sup>14</sup>C-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
<p>i) Amplification of Linear Templates encoding different SH2 proteins by two steps of PCR... more <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<sub>d</sub> value determination using Microscale Thermophoresis.</p
<p>(A) Detection of fluorescent ADAP in SDS gel (excitation 633 nm), (B) Coomassie stain, (... more <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
<p>(A) Titration of ADAP-P versus selected SH2 domains immobilized on streptavidin magnetic... more <p>(A) Titration of ADAP-P versus selected SH2 domains immobilized on streptavidin magnetic beads. (B) K<sub>d</sub> 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
<p>(A) Determined K<sub>d</sub> values of the interaction of phosphorylated ADA... more <p>(A) Determined K<sub>d</sub> 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
PLoS ONE, 2013
The characterization of phosphotyrosine mediated protein-protein interactions is vital for the in... more 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 (K d 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 K d 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 K d 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, K d 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 K d values for phosphotyrosine/SH2 interactions on a phosphoprotein level.
Organic & Biomolecular Chemistry, 2012
The bioorthogonal and chemoselective fluorescence labelling of several cell-free synthesized prot... more The bioorthogonal and chemoselective fluorescence labelling of several cell-free synthesized proteins containing a site-specifically incorporated azido amino acid was possible using different alkyne-functionalized Ru(II) bathophenanthroline complexes. We were able to achieve a selective labelling even in complex mixtures of proteins despite the fact that ruthenium dyes normally show a high tendency for unspecific interactions with proteins and are commonly used for total staining of proteins. Since the employed Ru complexes are extremely robust, photo-stable and highly sensitive, the approach should be applicable to the production of labelled proteins for single molecule spectroscopy and fluorescence-based interaction studies.
Angewandte Chemie International Edition, 2009
Analytical Biochemistry, 2014
Angewandte Chemie, 2009
Wir bedanken uns für finanzielle Unterstützung bei der Deutschen Forschungsgemeinschaft (DFG) im ... more Wir bedanken uns für finanzielle Unterstützung bei der Deutschen Forschungsgemeinschaft (DFG) im Rahmen des Emmy-Noether-Programms (HA 4468/2-1) und des SFB 765 sowie beim Fonds der Chemischen Industrie (FCI). Wir danken Dr. Dirk Schwarzer, Dr. mann und Wiebke Ahlbrecht für experimentelle Beiträge und sehr hilfreiche Diskussionen. Hintergrundinformationen zu diesem Beitrag sind im WWW unter http://dx.