Structural Studies of Amyloid-Forming Proteins and Peptides

This dissertation begins with my work on Aβ oligomers. I focus on a soluble oligomeric species of Aβ1-42, termed spherical particles. This species forms in vitro at physiological pH, is heterogeneous in size and does not show regular secondary structure in circular dichroism spectra. The formation of spherical particles and fibrils under various pH values was monitored as a function of time by negative-stain transmission electron microscopy (TEM). The results suggest that spherical particles are off-fibrillation-pathway. Furthermore, based on the observations from electron microscopy, we developed a method to prepare lyophilized spherical particles that can be applied in physiological experiments.;To investigate the structure of α-synuclein, I crystallized α-synuclein segments by fusing them to maltose-binding protein (MBP). From crystal structures of the fusions, we have been able to trace a virtual model of the first 72 residues of α-synuclein. Instead of a mostly α-helical conformation observed in a lipid environment, our crystal structures show alpha helices only at residues 1-13 and 20-34. The remaining segments are extended loops or coils. All of the predicted fiber-forming segments based on the 3D profile method are in extended conformation. We further show that the MBP fusion proteins with fiber-forming segments from α-synuclein can form fiber-like nano-crystals or amyloid-like fibrils. Hence, our structures reveal intermediate states during amyloid formation of α-synuclein.;In order to further explore the potential of using MBP as a carrier protein to facilitate crystallization of difficult protein targets, I worked with Arthur Laganowsky and Angela Soriaga. We developed a new crystallization methodology that combines the concept of synthetic symmetrization with the approach of engineering metal binding sites. In this method, double histidine or cysteine mutations are introduced on the surface of target proteins, generating crystal lattice contacts or oligomeric assemblies upon coordination with metals. Metal-mediated synthetic symmetrization greatly expands the packing possibilities of target proteins and therefore increases the chance of getting diffraction-quality crystals. To demonstrate this method, we designed various T4 Lysozyme (T4L) and maltose-binding protein (MBP) mutants and co-crystallized them with one of the three metal ions: copper (Cu2+), nickel (Ni2+ ) and zinc (Zn2+). The approach resulted in 17 new crystal forms.;My current research focuses on crystallizing amyloid-forming segments and exploring non-typical steric zipper structures. The peptide GVVAAC, derived from α-synuclein, fibrillizes much faster in oxidizing conditions containing Cu2+ than in reducing conditions containing TCEP (tris (2-carboxyethyl) phosphine), suggesting that disulfide bonds may be involved in amyloid core. In addition, crystal structure of KDWSFY, a peptide derived from β 2-microglobulin, has been solved, showing a steric zipper with β-strands non-perpendicular to the fiber axis. Yet KDWSFY fibers show a typical cross-beta diffraction pattern, suggesting that staggered β-strands can constitute the core of amyloid fibrils. More importantly, the staggered β-strands may suggest a critical link between the barrel-like oligomers and the fibrils.;The work embodied in this dissertation provides structural information for some amyloid-forming proteins and peptides, namely the conversion between Aβ oligomers and fibrils (Chapter 1), the polymorphism of Aβ fibrils (Chapter 2), the transition of α-synuclein from native state to amyloid state (Chapter 3), and a putative structural relationship between amyloid oligomers and fibrils based on crystal structure of a nontypical steric zipper (Chapter 5). All together, they demonstrate the heterogeneity and transiency of some amyloid species and suggest the molecular mechanism underlying the conversion between different species. (Abstract shortened by UMI.).