(467e) A Conformation-Switching Protein Probe for Detection of Alpha Synuclein Oligomers

Specific detection of biological analytes is critical in the development of diagnostics, biosensors and therapeutic strategies in various areas. Most biomolecular detection systems rely on molecular probes, which bind to target analytes usually in a structure-dependent manner. These probes often display cross-reactivity with other non-target molecules, particularly those structurally similar to target analytes. Moreover, many probe-mediated detection methods are not rapid-responsive but time-consuming and washing-intensive. This is due to the absence of a functional linkage between binding of probes to anaytes and generation of macroscopic signals. Therefore, development of a novel platform for the creation of conformation-selective, rapid-responsive probes should be invaluable yet challenging due to the lack of a well-established design principle. Such a platform also benefits the detection of structurally different, yet similar protein aggregates implicated in many amyloid diseases.

α synuclein (αS) is a 140-amino acid, structurally flexible, intrinsically disordered protein (IDP), and its aggregation is implicated in Parkinson’s disease (PD). αS monomers are irregularly structured. αS oligomers are soluble aggregates which may possess β sheet structures. αS oligomers can further aggregate to αS fibrils exhibiting cross β sheet structures which are similar, but not identical to β sheet conformations found in αS oligomers. It is widely accepted that β sheet-structured αS oligomers are the major toxic agents in PD, and specific detection of αS oligomers is quintessential to develop relevant diagnostic strategies.

In this study, motivated by Nature’s use of IDPs as conformation-switching biosensors, we engineered an αS variant, PG65, together with conformation-sensitive fluorescence to create a novel protein probe platform for rapid, specific and quantitative detection of αS oligomers. Our results suggest that PG65 was bound to β sheet-structured αS oligomers, and this binding event was functionally linked to fluorescence signaling. A linkage between binding and signaling could be tuned by binding-induced conformational changes of PG65, enabling selective detection of αS oligomers over unfolded αS monomers, structurally similar αS fibrils and other amyloid oligomers. Our strategy to engineer the structural flexibility of IDP represents a new paradigm for designing rapid-responsive, conformation-switching molecular probes for detection of specific protein forms mediated by not only binding to analytes but also its linkage to signaling.