The Paravastu Lab uses biophysical techniques, primarily solid-state NMR, paired with computational simulations to characterize and understand the assembly of disease-related amyloids and de novo peptide assemblies.
One of the hallmarks of neurodegenerative diseases, such as Alzheimer’s, Parkinson’s, and Huntington’s, is the formation and accumulation of amyloids. Amyloids are formed from misfolded proteins that aggregate and self-assemble into ordered nanostructures. These self-assembled structures are thought to disrupt cellular function and damage surrounding tissue. Therefore, structural characterization is critical to our understanding of how these structures form and how to develop targeted therapeutics.
Amyloid plaques observed in Alzheimer’s patients are composed of the protein amyloid-β assembled into β-sheet-rich nanofibers. While previous studies focused on the structure of these Aβ nanofibrils, recent studies suggest that oligomers are the primary toxic species by interacting with cell membranes and receptors. Aβ oligomers can form on-pathway to eventually produce fibers or off-pathway remaining size limited. Our collaborator Dr. Rosenberry at the Mayo Clinic has produced stable off-pathway oligomers that exhibit a uniform size composed of roughly 50 peptide strands. We employ solid-state NMR to investigate the 3D structure of these oligomers to better understand the mechanism underlying their toxicity and size-limited nature.
In certain forms of glaucoma, genetic mutations to the eye protein myocilin results in an increased propensity to form amyloid fibers. Studies from our collaborator Dr. Raquel Lieberman (GT) identified two major nanofiber morphologies, straight fibrils and circular fibrils, resulting from different assembly conditions. Through analysis of amyloidogenic regions of the full-length protein, two peptide sequences, P1 and P3, were determined to self-assemble and recapitulate these two distinct nanofiber structures. In collaboration with the Lieberman group, we are currently uncovering the molecular-level structure of these peptide nanofibers as well as fibers formed from the full-length protein. A deeper understanding of the mechanics leading to these nanofiber structures will aid in the development of therapeutics to treat this disease.
De Novo Peptide Assemblies
Insights into the biophysics resulting in disease-related amyloids spurred the design of de novo peptide sequences to form β-sheet and coiled-coil nanofibers. These peptide-based biomaterials have shown promise in a range of biomedical applications such as drug delivery, immunoengineering, and regenerative medicine. The Paravastu lab focuses on the evaluating the molecular-level organization of these peptides to gain insights into the sequence-to-structure relationships guiding the design of next-generation peptide assemblies.
Designer self-assembling β-sheet peptides
Designer self-assembling peptides are typically created by a rational choice of amino acid sequence patterning. By alternating hydrophobic and hydrophilic side chains, the peptide sequence dictates a β-strand secondary structure and an intermolecular organization into β-sheets. Using solid-state NMR and other biophysical measurement techniques, we have studied the self-assembly mechanism of the RADA16-I designer peptide, a synthetic amphipathic peptide known to self-assemble into nanofibers. By studying the structural changes that occurred during self-assembly, we were able to generate insights to improve RADA16-I functionality in supporting cell cultures and tissue regeneration.
Our lab is also taking a different approach in designing novel self-assembling peptides via a closed-loop design strategy. By integrating solid-state NMR experimental knowledge with computational structural design, we aim to examine the space of de novo peptide sequences more efficiently.
Designer coassembling coiled-coil peptides
Peptide coassembly emerged as a novel expansion to the forms and functions possible in these supramolecular biomaterials. One well-known design is the SAF peptides designed by Woolfson et al. which is composed of two distinct peptide sequences that fold into α-helices and cooperatively coassemble into a single nanofiber structure. The helical peptides designs utilize complementary charged patches on each end resulting in “sticky ends” that are electrostatically attracted to each other. Through solid-state NMR measurements, we observed an unusual temperature-dependent structural transition from the intended α-helical structure to β-sheets. Future work is focused on understanding peptides can be designed to controllably undergo this structural transition to access novel β-sheet structures.
Designer coassembling β-sheet peptides
Recently, coassembly of two distinct peptides into two-component β-sheet-rich nanofibers has been demonstrated. Compared to the vast literature on self-assembling peptides, only a handful of coassembling β-sheet peptide pairs are known. The majority of these designs rely on charge complementary sequences that are driven to coassemble by electrostatic attraction. Because of their charged nature, peptide strands were hypothesized to alternate perfectly along the β-sheet axis. However, solid-state NMR measurements and coarse-grained discontinuous molecular dynamics (DMD) simulations have shown a significant number of self-associated peptide pairs. Together with our collaborators Dr. Carol Hall (NC State) and Dr. Gregory Hudalla (UF), we apply a combined computational and experimental approach towards understanding the biophysics governing peptide coassembly vs self-assembly to inform the next-generation of coassembling peptide sequence designs.
DMD Image credit: Hall Group