Investigating the molecular basis of Menke Hennekam syndrome : a structural and functional analysis of the TAZ2 & IDR4 domains of CREB binding protein : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North, New Zealand
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2023
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Massey University
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Abstract
Menke Hennekam syndrome (MHS) is a rare genetic disorder characterised by intellectual disability and very specific physical abnormalities. MHS occurs as a result of de novo mutations to the cAMP response element binding protein binding protein (CREB-BP or CBP) gene. CBP is a master gene regulator protein, containing both intrinsic histone acetyltransferase activity and transcriptional coactivator activity. MHS associated mutations are located within exons 30 and 31 of the CBP gene, which encode the TAZ2 and IDR4 domains. The role which the TAZ2 and IDR4 domains play in the gene regulation carried out by CBP are yet to be fully understood. Previous research has established DNA binding activity by both TAZ2 and IDR4 domains, in addition to a lack of HAT specificity in the absence of the TAZ2 domain. It is suggested that the TAZ2 domain and neighbouring residues of the IDR4, may be involved in a regulatory interaction with DNA targets of the HAT domain. This study focused on uncovering more about the molecular basis of MHS by comparing the behaviour of MHS associated mutant CBP fragments of the TAZ2 and IDR4 domains with wild type fragments. Comparisons of structure, stability and DNA binding activity were carried out utilising circular dichroism spectroscopy, electrophoretic shift mobility assays and biolayer interferometry assays. The results produced throughout these experiments displayed that the MHS associated TAZ2-IDR4 mutant fragment contained DNA binding activity similar to the wild type TAZ2-IDR4 CBP fragments, during both EMSA and BLItz DNA binding assays. Structural investigations by circular dichroism spectroscopy of the MHS associated CBP mutants revealed a lack of native folding by the mutant protein fragments, which were prone to aggregation throughout expression and purification.
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