A conformation-switching peptide probe for rapid, specific, quantitative and sensitive detection of amyloid aggregates

New York University, New York NY

Investigators

Abstract

INTELLECTUAL MERIT. The goal of this project is to develop a conformation-switching peptide probe for rapid, specific, quantitative and sensitive detection of conformationally distinct yet similar analytes. Our target analytes are aggregates formed by a natively unfolded peptide, β amyloid (Aβ) implicated in Alzheimer?s disease (AD), known to form oligomeric and fibrillar assemblies. The heterogeneous Aβ aggregates formed during aggregation display different conformations and exert a varying extent of toxic effects. Thus, accurate and reliable detection of distinct Aβ aggregates represents a significant step toward a better understanding of the exact nature of Aβ aggregation and the development of early diagnostics as well as therapeutic drugs. Unfortunately, the existence of conformational similarity among distinct Aβ aggregates makes specific detection extremely difficult. Rapid, specific, quantitative and sensitive detection is particularly important yet currently unavailable in probing Aβ oligomeric aggregates, which are believed to be the major toxic agents in AD. In our previous studies, we developed a peptide probe, PG46, for rapid, specific and quantitative detection of Aβ oligomers. PG46 was created by integrating Aβ self-recognition sequences with the conformation-sensitive biarsenical dye, FlAsH. PG46 was found to specifically bind to Aβ oligomers and display an increase in FlAsH fluorescence upon such binding. Rapid, quantitative measurements of Aβ oligomers were also possible with PG46. Sensitivity for detection of Aβ oligomers was further improved by engineering of PG46. The sensitivity of our current peptide probes, though significant, is still not optimal. Peptide probes detecting other Aβ aggregates are also urgently needed for accurate profiling of Aβ aggregation. Moreover, understanding of the detection mechanism of our peptide probes is limited. A broader mechanistic understanding is critical for not only the refinement and optimization of our initial design principle to develop highly effective Aβ-specific probes, but also the extension of our strategy to create molecular probes for detection of amyloid aggregates formed by other natively unfolded proteins. The principal objective of the proposed project is to (1) create a novel class of peptide probes for highly specific and sensitive detection of distinct Aβ aggregates at physiological concentrations and (2) reveal the mechanism of detection of Aβ aggregates by our peptide probes. BROADER IMPACT. Our strategy to create peptide probes for Aβ detection represents a new paradigm for design of conformation-switching biosensors, motivated by Nature?s use of natively unfolded proteins as structure-switching biosensors for rapid and specific signaling processes. When successfully completed, our peptide probes will serve as biosensing tools to (1) advance understanding of the molecular basis of Aβ aggregation under physiologically relevant conditions, (2) identify, in a highthroughput assay, aggregation inhibitors/modulators targeting a specific aggregation step and, ultimately (3) establish correlations between Aβ aggregation profiles in biological samples and cellular/clinical manifestations of Aβ aggregation. Outcomes from our mechanistic studies are anticipated to provide an important foundation for (1) understanding of conformation-switching biosensors derived from natively unfolded proteins, (2) additional optimization of Aβ specific probes and (3) the creation of a biosensor for detection of other amyloid protein aggregates, implicated in > 20 protein misfolding diseases. The integration of research, education and outreach will result in the educational and professional development of K-12, undergraduate and graduate students as well as teachers in a multidisciplinary area that overlaps biochemistry, biophysics and protein engineering. The problem-driven learning through research projects, the biochemical engineering courses taught by the PI and an outreach effort through New York Hall of Science will foster students? creativity and problem solving ability. The proposed education and outreach plans tightly integrate with the research plan, and will focus on expanding the participation of underrepresented minority students in the study of science and engineering. The research results and educational materials will also be made available to the public in various formats including a video file on the PI?s web site.