Structural study of monomeric, oligomeric and fibrillar yeast prion protein Sup35

点击翻译按钮获取中文摘要

DESCRIPTION (provided by applicant): Prions are proteins that can switch to self-perpetuating, infectious conformations. The abilities of prions to replicate, form structurally distinct strains, and to establish and overcome transmission barriers between species are poorly understood. Using arrays of surface-bound peptides, small elements of primary sequence were found to control the switch to the prion state with exquisite specificity for the yeast prion Sup35. These same sequence elements govern the formation of distinct self-perpetuating conformations (prion strains) and determine species specific seeding activities for Sup35 from two different yeast strains. The amino acid compositon of these sequences elements is almost identical, but they do not cross-react. What are the structural differences in these regions that account for the species barrier and strain specificity? Structural investigation of prion proteins is often complicated by the fact that under native conditions, high concentration prion protein samples will spontaneously form fibers on (or faster than) the timescale of the experiment. To trap the monomeric form of NM for structural study, we will use mutant versions of NM with impaired aggregation kinetics, samples made in the presence of Hsp104 and encapusulation in reverse micelles in combination with segmental isotopic labeling and improved NMR instrumentation to interrogate the structure of the fiber nucleating regions of Sc and Ca NM. Antibodies that selectively recognize only the mature oligomeric species can keep solutions of oligomers stable indefinately. To isolate the oligomeric form of NM, we will raise a variety of single chain variable fragments (scFv) using yeast surface display for Sc and Ca NM. The structure of the peptides corresponding to the identified sequence elements of these proteins will be solved in complex with the unlabled scFv using solution NMR methods. To isolate the interesting structural elements of NM fibers, we will try to block or eliminate the polymerization interface of partially formed fibers. Amyloid protein aggregates have been increasingly implicated in human diseases, including prion-based encephalopathies, noninfectious neurodegenerative diseases and systemic amyloidoses. Soluble prion proteins form amyloid fibers via an on-pathway oligomeric intermediate that is thought to be the toxic species. Insight into the molecular structure of Sup35NM from two different species of yeast in the monomeric, oligomeric and fibers will help explain how one protein can switch between the prion and non-prion states and provide a structural rational for the species barrier for prion infection. Detailed structures of prion proteins will help inform drug design efforts against amyloid diseases.