In the Sohail Lab, we aim to gain physical insight into protein self-assembly, its role in human disease, and how to better control it for biomaterial applications. The core of our research uses Fluorescence Lifetime Imaging Microscopy (FLIM) to assess mixtures of fibrils with distinct morphologies at the single fibril level.
What are the chemical and physical driving forces of amyloid assembly?
The kinetics of amyloid formation for functional and disease-related amyloids have been shown to be strongly affected by the underlying polypeptide sequence and physical and chemical environmental conditions such as temperature, air-water interface, concentration, pH, and ionic strength. However, amyloid formation is dependent on complex protein-protein interactions, making it difficult to predict how these factors will affect amyloid formation for a particular construct. We use FLIM to assess the conformational heterogeneity of the underlying ensemble of fibrils formed under different conditions, by measuring the lifetimes of dyes that bind specifically to amyloid fibrils.
Amyloid-β 42
Amyloid-β 42 (Aβ42) is a 42 residue peptide associated with Alzheimer’s Disease. Aβ42 is intrinsically disordered in solution (no stable secondary structure), but ultimately forms amyloid fibrils at concentrations as low as hundreds of nanomolar. Aβ42 has a net charge of -3.2 at physiological pH and thus should be sensitive to pH changes and solution ionic strength. Aβ40 is the 40-residue isoform of Aβ. Aβ40 is more prevalent in the body, but has less of an association with disease and a significantly lower propensity to form amyloid fibrils in vitro. Polymorphs (structurally distinct fibrils) of Aβ have been well documented, but we hope to elucidate the physical molecular mechanics responsible for promoting different fibril structures.
PolyQ
Expanded polyglutamine (polyQ) tracts are associated with several neurodegenerative diseases, most notably, Huntington’s Disease. We use synthetic polyQ peptides (homopolymers of all glutamine residues) as a model system. PolyQ peptides of various lengths can form amyloid fibrils in vitro, with sequence length directly correlated with aggregation propensity. PolyQ offers an interesting, reductionist system to study the effects of sequence length on the amyloid structure since the sequences contain repeats of the same amino acid residue.
Assessing different fluorescent dyes for use in FLIM studies
Thioflavin T (ThT) is a fluorescent molecular rotor molecule with a high affinity for amyloid fibrils. In aqueous solution, ThT has a low quantum yield and fast (10s of picoseconds) fluorescent lifetime. However, upon binding to amyloid fibrils, there is a large increase in ThT quantum yield (fluorescence emission intensity) and a lengthening of the fluorescence lifetime to the nanosecond range. ThT is the gold standard dye for identifying amyloids, but there are many other dyes that bind specifically to amyloids through different mechanisms. We aim to understand how we can use a suite of fluorescent dyes to more fully understand underlying amyloid structure through changes in their fluorescence lifetimes upon binding to amyloids formed under different conditions.
CsgA (Curli)
Amyloids are most well known for their connection to neurodegenerative diseases, but they are also found as functional assemblies in all classes of life. Curli is the most well-studied functional amyloid and is found in bacterial biofilms. CsgA is the primary amyloid-forming subunit of Curli. We are interested in how peptide fragments of CsgA form amyloid under various environmental conditions. We seek to understand the differences in assembly and structure of this functional amyloid when compared to its disease-related counterparts since the primary cross-β amyloid structure is conserved.
Lysozyme
Hen egg white lysozyme (HEWL) is a crucial enzyme with the primary role of preventing bacterial infections. In vitro HEWL can form amyloid fibrils under diverse conditions. HEWL is an excellent model system to test out different fluorescent dyes in our FLIM studies and offers a well-documented example of pH-dependent fibril polymorphism.