Isothermal Titration Calorimetry and Vesicle Leakage Assays Highlight the Differential Behaviors of Tau Repeat Segments Upon Interaction with Anionic Lipid Membranes

Abstract

Alzheimer's Disease (AD) is the sixth-leading cause of death in the United States and is due to protein misfolding. One of the proteins implicated in this misfolding is Tau, which is a protein that stabilizes the cytoskeletal structure in neurons. Tau contains four repeat segments (R1-R4) that are crucial to its function, but are also the site of the misfolding that forms neurofibrillary tangles (NFTs). These NFTs inhibit signals between neurons and invokes processes which cause degradation of the neuronal cell. A number of previous studies reveal that anionic membranes interact with tau and induce misfolding of the protein. Here, we have investigated how each repeat segment of tau interacts with anionic and neutral membranes using isothermal titration calorimeter (ITC) and leakage assays. We have determined the equilibrium binding constants for peptide: membrane interactions, along with the thermodynamic parameters ΔH, ΔS, and ΔCp. We found that R2 and R3 repeat segments interact more favorably with anionic membranes, and none of the segments interacted strongly with neutral membranes. The differential behaviors of these sequences was also evident in their driving forces to membrane binding - both classical and nonclassical hydrophobic effects were observed among these sequences. Leakage assays showed that all sequences display lytic activity to anionic membranes but not neutral membranes. Taken together, these results align with models of NFT formation that invoke misfolding events at the membrane surface. Further exploration will include experiments using NMR to see how the phosphates on the side chains of the amino acids play a role in the structural composition of the repeat segments.