Unraveling the Molecular Complexity of N-Terminus Huntingtin Oligomers: Insights into Polymorphic Structures.

Huntington's disease (HD) is a fatal neurodegenerative disorder resulting from an abnormal expansion of polyglutamine (polyQ) repeats in the N-terminus of the huntingtin protein. When the polyQ tract surpasses 35 repeats, the mutated protein undergoes misfolding, culminating in the formation of intracellular aggregates. Research in mouse models suggests that HD pathogenesis involves the aggregation of N-terminal fragments of the huntingtin protein (htt). These early oligomeric assemblies of htt, exhibiting diverse characteristics during aggregation, are implicated as potential toxic entities in HD. However, a consensus on their specific structures remains elusive. Understanding the heterogeneous nature of htt oligomers provides crucial insights into disease mechanisms, emphasizing the need to identify various oligomeric conformations as potential therapeutic targets. Employing coarse-grained molecular dynamics, our study aims to elucidate the mechanisms governing the aggregation process and resultant aggregate architectures of htt. The polyQ tract within htt is flanked by two regions: an N-terminal domain (N17) and a short C-terminal proline-rich segment. We conducted self-assembly simulations involving five distinct N17 + polyQ systems with polyQ lengths ranging from 7 to 45, utilizing the ProMPT force field. Prolongation of the polyQ domain correlates with an increase in ß-sheet-rich structures. Longer polyQ lengths favor intramolecular ß-sheets over intermolecular interactions due to the folding of the elongated polyQ domain into hairpin-rich conformations. Importantly, variations in polyQ length significantly influence resulting oligomeric structures. Shorter polyQ domains lead to N17 domain aggregation, forming a hydrophobic core, while longer polyQ lengths introduce a competition between N17 hydrophobic interactions and polyQ polar interactions, resulting in densely packed polyQ cores with outwardly distributed N17 domains. Additionally, at extended polyQ lengths, we observe distinct oligomeric conformations with varying degrees of N17 bundling. These findings can help explain the toxic gain-of-function that htt with expanded polyQ acquires.

Opposing roles of organic salts on mini-protein structure.

We investigated the effects of 1-ethyl-3-methylimidazolium chloride ([EMIM][Cl]) and choline chloride ([Chol][Cl]) on the local environment and conformational landscapes of Trp-cage and Trpzip4 mini-proteins using experimental and computational approaches. Fluorescence experiments and computational simulations revealed distinct behaviors of the mini-proteins in the presence of these organic salts. [EMIM][Cl] showed a strong interaction with Trp-cage, leading to fluorescence quenching and destabilization of its native structural interactions. Conversely, [Chol][Cl] had a negligible impact on Trp-cage fluorescence at low concentrations but increased it at high concentrations, indicating a stabilizing role. Computational simulations elucidated that [EMIM][Cl] disrupted the hydrophobic core packing and decreased proline-aromatic residue contacts in Trp-cage, resulting in a more exposed environment for Trp residues. In contrast, [Chol][Cl] subtly influenced the hydrophobic core packing, creating a hydrophobic environment near the tryptophan residues. Circular dichroism experiments revealed that [Chol][Cl] stabilized the secondary structure of both mini-proteins, although computational simulations did not show significant changes in secondary content at the explored concentrations. The simulations also demonstrated a more rugged free energy landscape for Trp-cage and Trpzip4 in [EMIM][Cl], suggesting destabilization of the tertiary structure for Trp-cage and secondary structure for Trpzip4. Similar fluorescence trends were observed for Trpzip4, with [EMIM][Cl] quenching fluorescence and exhibiting stronger interaction, while [Chol][Cl] increased the fluorescence at high concentrations. These findings highlight the interplay between [EMIM][Cl] and [Chol][Cl] with the mini-proteins and provide a detailed molecular-level understanding of how these organic salts impact the nearby surroundings and structural variations. Understanding such interactions is valuable for diverse applications, from biochemistry to materials science.

A Simple Radioassay to Detect Nanoscale Membrane Disruption.

Understanding the mechanisms and kinetics of membrane damage is of interest to researchers in several overlapping fields of biology. In this study, we describe the development and validation of a simple 32PO43- release radioassay used to track nanometer-scale damage to the bacterial cell membrane. Nanoscale membrane damage will result in the release of small cytoplasmic molecules, such as amino acids, sugars, and osmolytes. Our radioassay tracks the release of these molecules using the release of cytoplasmic 32PO43- as a proxy. Our assay can both detect 32PO43- release and track release kinetics in the order of minutes. We demonstrate the use of our radioassay using A. baumannii treated with colistin and Ω76: two agents known to cause membrane damage. Our assay tracks greater membrane damage in A. baumannii treated with both these agents, compared to an untreated control. Our assay fills a niche that is not covered by traditional 51Cr release radioassays and fluorescent staining techniques. Furthermore, our assay can potentially be used to track membrane damage in other membrane systems such as lipid vesicles, animal cells, and organelles.

Ω76: A designed antimicrobial peptide to combat carbapenem- and tigecycline-resistant Acinetobacter baumannii.

Drug resistance is a public health concern that threatens to undermine decades of medical progress. ESKAPE pathogens cause most nosocomial infections, and are frequently resistant to carbapenem antibiotics, usually leaving tigecycline and colistin as the last treatment options. However, increasing tigecycline resistance and colistin's nephrotoxicity severely restrict use of these antibiotics. We have designed antimicrobial peptides using a maximum common subgraph approach. Our best peptide (Ω76) displayed high efficacy against carbapenem and tigecycline-resistant Acinetobacter baumannii in mice. Mice treated with repeated sublethal doses of Ω76 displayed no signs of chronic toxicity. Sublethal Ω76 doses co-administered alongside sublethal colistin doses displayed no additive toxicity. These results indicate that Ω76 can potentially supplement or replace colistin, especially where nephrotoxicity is a concern. To our knowledge, no other existing antibiotics occupy this clinical niche. Mechanistically, Ω76 adopts an α-helical structure in membranes, causing rapid membrane disruption, leakage, and bacterial death.