The architecture and assembly of fibrillin microfibrils, key components of elastic fibres. (360G-Wellcome-086613_Z_08_Z)

In this application we will investigate the architecture and assembly of elastic fibre microfibrils, targeting key fragments of fibrillin involved in microfibril assembly. Specifically we aim to i) Use NMR to identify the individual three-dimensional structure and dynamic properties of an N-terminal fragment (Nterm-EGF3) which contains the C-terminal interaction site. In parallel a homology model of cbEGF41-43 will be built based on the known structures of fibrillin-1 cbEGF domains and NMR wil l be used to confirm the predicted rod-like structural arrangement of the construct by the identification of conserved interdomain packing interactions ii) Identify the minimal interacting N- and C-terminal complex by fine mapping of Nterm-EGF3 and cbEGF41-43, determine its stability by surface plasmon resonance (SPR) and precisely define the interacting surfaces by NMR shift mapping and/or crystallography iii) Identify the dynamic properties and pairwise interactions of the first hybrid domain (hyb1) and adjacent domains EGF3 and cbEGF1 iv) Use cellular models of matrix assembly combined with structure-informed mutagenesis to test the importance of putative interface residues for N- and C-terminal complex formation .