Probing the role of helix-1 in amyloid formation by serum amyloid A



 DESCRIPTION (provided by applicant): AA amyloidosis is a severe complication of chronic inflammatory disorders and is potentially fatal. The amyloid fibrils involved in AA amyloidosis are derived from serum amyloid A (SAA) which is an acute phase reactant protein. In AA amyloidosis, circulating amyloid fibrils are deposited in organs ultimately leading to their failur. Serum amyloid A (SAA) is the major protein component of amyloid fibrils associated with AA amyloidosis. Recent molecular dynamics simulations have provided an in silico model for SAA misfolding in which the N-terminal helical region (Helix-1) of the molecule plays a pivotal role. This portion of the polypeptide is believed to transition from a α-helix to β-hairpin. The β-haipin units are postulated associate and stack upon themselves to produce amyloid fibrils. It is the focus of this proposal to experimentally investigate this model of SAA misfolding through the synthesis and characterization of peptide analogs of the helix-1 region. The roles of specific amino acids will be probed and their trajectory during SAA misfolding interrogated. Peptides containing specific point mutations along with isotopically labeled residues will be employed in conjunction with thioflavin T kinetic aggregation assays, FRET, Raman and IR vibrational spectroscopic studies to determine the key molecular interactions and mechanism responsible for SAA fibrillization. Transmission electron microscopy will also be employed to determine the morphology of amyloid fibrils. Experimental data will be compared and contrasted to the in silico model. One of the major goals of this project is to provide underrepresented minority students, >50% of our student body, with the opportunity to conduct research and gain experience working in a modern chemical laboratory. Students involved in this project will be exposed to a variety of techniques ranging from solid phase peptide synthesis and kinetic assays to advanced spectroscopic and biochemical methods. This project provides students the opportunity to apply theories learned in the class room to real world chemical problems in the laboratory. Hands-on research experience is one of the most effective approaches for the development of critical thinking and improving the scientific infrastructure of the United States o America. Moreover, research experiences that are based on current technology allow students to solidify and build on their knowledge in ways that enhances a connection to the real world. This experience will help prepare students for graduate programs and employment in the chemical sciences. The educational impact of this project also extends to local high school and community college students already working in the PIs laboratories. These high school and community college students benefit from their direct collaboration with NIH-AREA trainees and by attending all scholarly activities of research groups, such as seminars, conferences and journal clubs.