(161c) Co-Assembling Oppositely Charges Peptides for Salt Bridge Analysis

Background: There are 5,7 million Alzheimer’s, 1 million Parkinson diseases patients in the U.S.¹ The most noxious mutations of pathological fibrillar protein aggregates, such prion, amyloid-b, and a-synuclein fibers, have one common feature; increase of salt bridges. These salt bridges increase their ability to form pathological stage to initiate Prion, Alzheimer’s, and Parkinson diseases; none of which have known cures. Despite such importance, the effects of salt bridges on forming and stabilizing the fibrillar aggregates have not been studied in detail by peptide engineers. Understanding the effects of salt bridges on the structure and properties in systematic way by mimicking the key pathological mutations of fibrillar proteins, then correlating them to the functionalities of the new structures, can be an important step for finding a cure for these diseases. We aim to understand the effects of salt bridges on peptide assembly into one-dimensional structures to develop concomitant strategies for engineering biomimetic materials with various properties. Here, we show a new design of Co-assembling Oppositely-charged Peptides (CoOP) to study the effects of salt bridges on peptide self-assembly mechanism.

Methods: CoOP is a hexapeptide system, each peptide has two charges on both ends and a hydrophobic core. In this study, we explored the effects of salt bridges between Lysine (K), Glutamic acid (E), Arginine (R), and Aspartic acid (D) on different CoOP systems, with various hydrophobic amino acids in the core. We integrated computational methods into analyses, instead of prediction, of the assembly mechanism. Atomic-resolution molecular dynamics (MD) simulations used to probe the free energy of association and probability of amino acid contact during co-assembly. The effects of the substitution domain on both the assembly kinetics were investigated via pyrene and Congo red staining. We used FTIR and circular dichroism to understand the secondary structures and identified the physical and mechanical properties of the emergent materials via TEM, AFM, and rheology. We combined the results of these approaches to understand the intermolecular association on assembly kinetics, structural and mechanical properties of the end product.

Results: Salt bridges not only assist with aqueous solubility, but also provide a relatively long-ranged attractive force to encourage peptides to come together. The accessible surface hydrophobic area of the amino acids defined the favorably of their assembly, while the initial interactions happen between the electrostatic groups. The results of experimental kinetics measurement are found correlated to the distances between the interacting peptides and the thermodynamic free energy measurements, and thus, the experimental and computational analyses are integrated to explain the complex assembly kinetics, which is an advantage of simple design of the CoOP system. Importantly, we showed an undisrupted hydrophobic core is an important parameter for establishing the assembly in CoOP, while charges around the hydrophobic region enhance the stability.

Understanding salt bridges can significantly advance our understanding on diseases causing by fibrillar diseases. The effects of salt bridges on the structural and mechanical properties of peptide-based materials in different conditions will broaden their application as nanomaterials with desired properties. Importantly, the library of structural properties with functionalities on cells can be an important prediction tool for identifying the peptide sequence for a desired tool; can pave the way for materials genomics.

(1) Alzheimer’s Disease: Get The Facts https://www.usagainstalzheimers.org/alzheimers-disease-get-facts (accessed Apr 12, 2021).