C1, SAP and ZiCo: structural studies of three metal-binding proteins from a crystallographic perspective

Atomic resolution models of proteins are crucial for understanding their biological mechanisms and provide insights into the relationship between protein sequence and structure. Many protein structures incorporate metal ions, exploiting their unique chemistry as reaction centres or for structural stability. This thesis describes the progress made towards solving the structures of three such metal-binding proteins by means of X-ray crystallography. Complement component C1 is a large protein complex that initiates the first line of immune defence and requires calcium for structural stability. Fragments of C1 have already been solved at high resolution, but there are no accurate models of the assembled complex. In this work, a new method for purifying intact C1 from human serum was developed and the purified complex was characterised by various methods. Finally, attempts were made to crystallise native human C1 with a view to obtaining high-resolution structures of the entire complex. Serum amyloid P is another serum protein, also thought to be involved in the immune response. It is often found associated with amyloid deposits, although SAP binds a variety of ligands in a calcium-dependent manner. While the structure of SAP has been determined, its physiological function is still not fully understood. SAP was purified using established methods and its ligand-binding properties were investigated under various conditions using dynamic light scattering, in an attempt to gather more information about the possible function of this molecule. Finally, ZiCo is a small peptide that was designed to switch between a multimeric coiled coil and a monomeric zinc finger fold on binding zinc. The system has been characterised extensively in solution, but high-resolution structures are required to validate the design. ZiCo was crystallised and diffraction data were collected. The structure of the peptide was partially solved, indicating that the multimeric form of the ZiCo peptide is indeed a trimeric coiled coil.