| Structural studies of proteins is an active area of research that has grown steadily over the last 60 years. The number of proteins with structural solutions has increased due to the continual development of techniques and methods that overcome earlier limitations in protein purification and crystallization and also diffraction methods. The main thrust of this thesis is to present a novel approach developed to stabilize and crystallize proteins containing a coiled-coil motif. The divided and conquer mentality in crystallography suggests that if a complete protein is not amenable to study it can be broken down and studied in smaller parts. A coiled-coil region that is found dividing globular domains can be an ideal region for separation of these globular domains with minimal interference to their function. However, dividing a protein at the coiled-coil can weaken the oligomeric propensity that may be necessary for correct function of the globular domains. Traditionally the leucine zipper from the transcription factor GCN4 has been used to stabilize artificially truncated coiled-coils. In this thesis, the bacteriophage &phis;29 scaffolding protein Gp7, the human DNA repair ligase protein Xrcc4, and the microtubule associated protein EB1, were successfully used to stabilize and crystallize the coiled-coil containing spindle pole body protein Cnm67 and the overlap complex of smooth muscle tropomyosin. These "stopper constructs" have the advantage of containing different sized globular domains in addition to a coiled-coil region used for fusion to the target protein. These globular domains can be tailored to increase the symmetry of the final construct, potentially leading to enhanced crystallization characteristics. |
