Amy Ruschak
From WikidChem
Amy is a graduate student in the Miranker Lab .
Research
The spontaneous self-assembly of proteins into amyloid fibers is not only associated with disease but is a general structural state that can be formed by most any polypeptide chain. Self-assembly of beta–sheet amyloid structure occurs by a nucleation dependent mechanism that gives rise to a characteristic sigmoidal reaction profile. Fiber formation pathways involve an intermediate known as the nucleus, which is the highest energy state in assembly. Amyloid fiber formation is rate-limited by stabilization of the nucleus by an irreversible oligomeric and/or conformational change in a kinetic step known as nucleation. Structural determination of the nucleus is difficult due to its exceedingly low population.
The goal of my project is to determine the structure of the nucleus and the nature of the nucleation step of fiber formation by a peptide derived from residues 20-29 of the diabetes-related protein, islet amyloid polypeptide (IAPP). First, we determined the overall topological arrangement of peptides within the fiber using a variety of techniques including electron paramagnetic resonance spectroscopy (EPR), crosslinking experiments, and mutagenesis. Next, we used kinetics to determine (1) the size of the nucleating species (2) the energy changes associated with nucleation (3) the features of the fiber structure that are present within the nucleating species.
We determined that fibers are composed of parallel, in-register beta–sheets, and the amyloid nucleus is composed of a single, two-stranded beta–sheet. We have modeled the nucleation step to be the product of collision of two such nuclei to form the inter-sheet interface of the fiber. The nucleation process is catalyzed by surfaces including the surface of the fiber.
