Abstract 2307 Evaluating the thermal stability of congenital heart diseases-associated TBX5 missense mutants

Transcription factors (TFs) form protein-DNA complexes that regulate gene expression and control most biological processes, such as proper heart development in the case of TBX5. Mutant TBX5 has been associated with congenital heart diseases (CHDs), which affect approximately 1 in every 100 births. However, a molecular mechanism that defines causality remains unknown. We hypothesize that missense mutations will change the thermal stability of cardiac TFs. A protein's melting temperature (Tm) describes the temperature at which half of the protein in the solution is unfolded, and the other half is in its native (functional) state. Tm values can be used to measure a protein's relative stability, with higher values indicating greater stability and vice versa. This project aims to determine changes in the Tm in CHD-associated mutations of the TBX5 DNA-binding domain (T-Box domain). Towards this goal, wild-type (wt) TBX5 T-box domain and seven missense mutants (I101F, I54T, M74V, G80R, R113K, R273Q, R237W) were cloned, expressed, and purified through Ni-NTA affinity chromatography. All eight recombinant T-box domains have previously been demonstrated to have differential TF-DNA binding affinity for the TBX5 putative binding site. To further understand changes in mutant TF-DNA bindings, we will evaluate changes in thermal stability for all seven mutants and the wt T-box domain through Differential Scanning Fluorimetry (DSF). Samples were evaluated in a temperature range from 25°C to 95°C with a ramp of 0.05°C/s, causing the T-box domains to unfold and expose hydrophobic regions where the SYPRO ORANGE dye can bind and emit a fluorescent signal. Through this approach, we obtained an average Tm of 55.69°C for the TBX5 T-Box domain. Our preliminary results show that the TBX5 I101F mutant shows a change in the average Tm value to 57.95°C (ΔTm = 2.26°C). When correlating this to the previously validated change in DNA-binding affinity, we found that the I101F loses the capacity to bind the putative binding site for the wt protein. Future steps consist of evaluating all the mutants and correlating their change in thermal stability with changes in DNA-binding affinities. Our findings will contribute to understanding the molecular mechanisms behind TBX5 mutants associated with CHDs by determining changes in the TF-DNA complex stability. Project supported by NIH-SC1GM127231, , University of Puerto Rico Rio Piedras Institutional Funds (FIPI), Puerto Rico Science, Technology, and Research Trust, and NIH Institutional Development Award (IDeA) INBRE (P20GM10347), NIH RISE (5R25GM061151–20), NSF: BioXFEL (STC-1231306), HRD-2008186, REU: PR-CLIMB (2050493 ), EPSCoR (1736026) & NIH:URISE (1T34GM145404-01A1).