Protein Expression, Purification, and Binding Assays to Characterize CSL Point Mutations Causing Adams-Oliver Syndrome
Start Date
1-8-2024 11:00 AM
End Date
1-8-2024 11:15 AM
Location
ALT 211
Abstract
Adams-Oliver Syndrome (AOS) is a rare multiple-malformation genetic disorder which is characterized by scalp, cranium, and transverse limb developmental defects. Several point mutations within the CSL transcription factor protein of the Notch signaling pathway have been associated with AOS. CSL can bind to DNA and transcriptional corepressor/coactivator proteins. Of the AOS-associated CSL point mutations tested, all have shown a significant reduction in DNA binding affinity while having no effect on CSL binding affinity to corepressors or coactivators. The following four point mutations in CSL were characterized: Y60C, R65G, S332R, and F66V. We purified each of these CSL variants, and performed thermal shift assays and isothermal titration calorimetry (ITC). The results indicate that each variant was properly folded but specifically showed reduced binding to DNA, agreeing with the previously published model of AOS. Future ITC experiments will be done to confirm that these CSL variants still bind to corepressors and coactivators.
Protein Expression, Purification, and Binding Assays to Characterize CSL Point Mutations Causing Adams-Oliver Syndrome
ALT 211
Adams-Oliver Syndrome (AOS) is a rare multiple-malformation genetic disorder which is characterized by scalp, cranium, and transverse limb developmental defects. Several point mutations within the CSL transcription factor protein of the Notch signaling pathway have been associated with AOS. CSL can bind to DNA and transcriptional corepressor/coactivator proteins. Of the AOS-associated CSL point mutations tested, all have shown a significant reduction in DNA binding affinity while having no effect on CSL binding affinity to corepressors or coactivators. The following four point mutations in CSL were characterized: Y60C, R65G, S332R, and F66V. We purified each of these CSL variants, and performed thermal shift assays and isothermal titration calorimetry (ITC). The results indicate that each variant was properly folded but specifically showed reduced binding to DNA, agreeing with the previously published model of AOS. Future ITC experiments will be done to confirm that these CSL variants still bind to corepressors and coactivators.
