CEEPC/IPM/CMSC - Abstrakt prezentace

(Česká konference hmotnostní spektrometrie 2019 - ThS-010)
Native nanoelectrospray-MS as a tool for quick evaluation of protein-DNA complex formation

Růžena Lišková 1,2 *, Jan Fiala 1,2, Daniel Kavan 1,2, Karel Vališ 1,2, Petr Novák 1,2

  1. Institute of Microbiology, CAS, Vestec
  2. Faculty of Science, Charles University, Prague

Abstrakt

Transcription factors mediate gene expression regulation through interactions with DNA and other regulatory proteins. Therefore, they play a crucial role in many biological processes including growth and development of organisms, various metabolic pathways or tumorigenesis. Due to their aforementioned properties, strict regulation of transcription factor’s activity is required and one of the possible ways how they are regulated, is through differences in their affinity to different DNA sequence motives. In our work, we have studied the effect of orientation and surrounding sequence of the DNA response motif on interaction of DNA binding domain (DBD) of transcription factor TEAD1 with its DNA response M-CAT motifs originating from regulatory regions of human genes.

First of all, we needed to check the recombinantly prepared TEAD1-DBD’s ability to form complexes with all the selected DNA oligonucleotides and to estimate Kd of each complex. Since methods usually used for protein-DNA Kd determination (such as thermophoresis, fluorescence anisotropy or gel shifts) are tedious and sample consuming or need one of the complex components to be labelled, they are not suitable for evaluation of multiple complexes in short time. Thus, we have tested the potential of native nanoelectrospray ionization coupled to FT-ICR MS for Kd determination.

Using six TEAD1-DBD∙M-CAT complexes with different Kd's that were previously determined by fluorescence anisotropy-based binding assay, we have observed all components (free DNA, free protein and the complex) in the obtained spectrum. Furthermore, the ratio of signal intensities of TEAD1-DBD in its free and complexed form was used to calculate bound fraction of TEAD1-DBD which was in agreement with known dissociation constants.

* Korespondující autor: ruzena.liskova@biomed.cas.cz

Poděkování:

This work was supported by the Charles University Grant Agency (1618218), the Czech Science Foundation (grant numbers 16-24309S), the Ministry of Education of the Czech Republic (programs “NPU II” project LQ1604 and OPVVV CZ.02.2.69/0.0/0.0/16_027/0007990).


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