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ProteoStat® Dye Mechanism
Selected Literature
ProteoStat® Protein Aggregation Assay Flyer
Proposed Signal Generation Mechanism
Figure 1: Thioflavin T - An early prototype molecular rotor dye used by Enzo scientists in the conceptual design of the ProteoStat® aggregate sensing dye. A family of 130 molecular rotor dyes was synthesized, each member of which was designed to inherently decrease its non-radiative decay rate and increase fluorescence emission upon binding to the surface of aggregated proteins.
β-rich peptide self-assemblies, arising from protein aggregation, typically adopt a cross-β sheet structure, characterized by an extended β-conformation, with the strands running perpendicular to the long axis of the assembly and with two or more β-sheet layer structures uniting together to form a stacked laminate-like structure, similar to plywood. Both parallel and anti-parallel β-strand orientations generate a specific arrangement of side chains in these self-assemblies, referred to as cross-strand ladders. These cross-strand ladders are comprised of repeating side-chain interactions running across β-strands within a β-sheet layer. The cross-strand ladders run parallel with the long axis of the assembly and arise from the inherently repetitive nature of self-assembly. Neighboring rows of cross-strand side chains occur regardless of peptide sequence, and form extended channel-like motifs along solvent-exposed surfaces of the assembly into which certain dye molecules can potentially bind. Aromatic amino acid residues, such as tyrosine, lining these channels, may promote high affinity dye binding. The described structural motif has been identified in a wide range of peptide and protein aggregates, being fundamental to the oligomeric aggregate and present in both amyloid fibers and amorphous structures.
In solution, when molecular rotation is not constrained, excited molecular rotor dye molecules apparently undergo a torsional relaxation which effectively competes with the radiative transition, rendering them essentially non-fluorescent. Simply stated, molecular rotor dyes are basically non-fluorescent due to free rotation around the central carbon-carbon single bond separating the different aromatic portions of the molecule (Figure 1). A dramatic increase in quantum yield occurs upon binding of the dye to the aggregated protein structure, arising from inhibition of the free rotation of its constituent aromatic rings (Figure 2). When the molecular rotor dye locks into the quaternary structure of protein aggregates, it becomes highly fluorescent, much in the same manner that ethidium bromide does when it intercalates into the three-dimensional structure of DNA.
Figure 2: Dye interaction with the quaternary structure of a protein aggregate. Adjacent cross-strand side-chain ladders of a self-assembled antiparallel β-sheet of the protein aggregate is depicted. The sheet backbone is highlighted as N and C residues flanking the α-carbon. Side chains are indicated by the letter R within a circle. Binding to this common structural motif, present in aggregated protein, restricts dye rotation around the central carbon-carbon single bond separating the different aromatic portions of the molecule. Two possible dye binding orientation are depicted as red arrows in the diagram, though binding at atomic level resolution has yet to be fully delineated .
