Biology subjects

Newberry, R. W.

Publications and source records attributed to Newberry, R. W..

5 recordsLinked to original sources

bioRxiv2026

Backbone Thioamide Substitution Enhances the Activity of Short Peptides in Modulating the Aggregation of α-Synuclein

The non-amyloid-{beta} component (NAC) region of the Parkinson's-associated protein -synuclein plays a key role in its pathogenic aggregation, motivating the development of molecules that target this critical region. Here, we show that a minimal NAC-derived motif, 66VGGAVVT72, can be reprogrammed through backbone engineering to modulate -synuclein aggregation. Backbone thioamide substitution of this peptide enhances its interactions with -synuclein fibrils and accelerates aggregation, whereas N-methylation disrupts {beta}-sheet hydrogen bonding and inhibits fibrillization. Strikingly, combining these modifications yields hybrid peptides that inhibit the fibrillization of full-length -synuclein at sub-stoichiometric concentrations. Consistent with in vitro results, these backbone-modified peptides can also reduce seeded -synuclein aggregation in cells. These results establish that minimal amyloidogenic sequences can be systematically tuned from aggregation promoters to inhibitors through backbone-level perturbations, particularly thioamide incorporation.

biochemistry↗

bioRxiv2026

Mutational Scanning of α-Synuclein using a Clickable Protein Tag Reveals Determinants of Membrane-Induced Aggregation

The cellular environment plays a critical role in shaping protein conformations, including aggregated states implicated in disease. One challenge to studying this relationship is that most techniques offering high-resolution insight into the nature of these aggregates cannot be deployed in living cells. Systematic mutagenesis presents an opportunity to bridge this gap but requires general and robust methods to detect protein aggregation across large numbers of variants. Here, we use clickable protein tags to generate FRET pairs in situ that can report protein aggregation in high throughput in living cells. We applied this strategy to probe the nature of cellular inclusions of -synuclein in a popular yeast model. Our results demonstrate that cellular aggregates of -synuclein in yeast are likely dominated by protein-membrane interactions, making the aggregation pathway in this cellular model very different than in many in vitro experiments. Furthermore, our comprehensive mutational data reveal the molecular determinants of membrane-induced aggregation. For example, residues that control membrane affinity have a profound effect on membrane-induced aggregation both in vitro and in cells. Furthermore, we discovered that glycine residues, particularly in the central region of the protein, act as gatekeepers to reduce membrane-induced aggregation. Mutational scanning with a clickable protein tag therefore provides high-resolution insights into cellular protein aggregates.

biochemistry↗

bioRxiv2025

Concentration-Dependent Mutational Scanning Probes the Cellular Folding Landscape of α-Synuclein in Yeast

The misfolding and aggregation of -synuclein is a central molecular event in the etiology of Parkinsons disease and related disorders. -Synuclein misfolding and pathology are both concentration-dependent, but it is not clear precisely how changes in concentration alter the folding landscape within cells. Whereas most conventional structural biology approaches offer limited resolution in living systems, deep mutational scanning can offer insight into the folding state of a protein in living cells, and we apply this method to probe concentration-dependent changes in the folding of -synuclein in a popular yeast model of pathology. We discover that at a wide range of cellular concentrations, -synuclein is highly biased toward formation of a membrane-bound amphiphilic helix that imparts toxicity. Population of this toxic state can be disrupted by mutations that reduce membrane affinity, which shift the folding equilibrium away from the membrane-bound state. Reduced-affinity variants exhibit distinct sensitivity to concentration relative to variants with WT-like affinity, likely because these variants are expressed at concentrations closer to their dissociation constant for membrane binding. These results show how mutational scanning can provide high-resolution insights into the folding landscape of proteins in living cells, which is likely to be of special utility for studying proteins that misfolding and/or aggregate. Impact StatementProtein misfolding is often concentration-dependent, but studying concentration-dependent changes in folding in living cells is challenging. By using high-throughput mutagenesis, we reveal changes in the population of toxic conformations of the Parkinsons-associated protein -synuclein. We discover that in a yeast model of pathology, -synuclein is highly biased toward membrane binding, which in turn disrupts cellular homeostasis.

biochemistry↗

bioRxiv2023

Liquid-liquid phase separation of alpha-synuclein is highly sensitive to sequence complexity

The Parkinsons-associated protein -synuclein (-syn) can undergo liquid-liquid phase separation (LLPS), which typically leads to the formation of amyloid fibrils. The coincidence of LLPS and amyloid formation has complicated the identification of the molecular determinants unique to LLPS of -syn. Moreover, the lack of strategies to selectively perturb LLPS makes it difficult to dissect the biological roles specific to -syn LLPS, independent of fibrillation. Herein, using a combination of subtle missense mutations, we show that LLPS of -syn is highly sensitive to its sequence complexity. In fact, we find that even a highly conservative mutation (V16I) that increases sequence complexity without perturbing physicochemical and structural properties, is sufficient to reduce LLPS by 75%; this effect can be reversed by an adjacent V-to-I mutation (V15I) that restores the original sequence complexity. A18T, a complexity-enhancing PD-associated mutation, was likewise found to reduce LLPS, implicating sequence complexity in -syn pathogenicity. Furthermore, leveraging the differences in LLPS propensities among different -syn variants, we demonstrate that fibrillation of -syn does not necessarily correlate with its LLPS. In fact, we identify mutations that selectively perturb LLPS or fibrillation of -syn, unlike previously studied mutations. The variants and design principles reported herein should therefore empower future studies to disentangle these two phenomena and distinguish their (patho)biological roles.

biochemistry↗

bioRxiv2020

Robust Sequence Determinants of α-Synuclein Toxicity in Yeast Implicate Membrane Binding

Protein conformations are shaped by cellular environments, but how environmental changes alter the conformational landscapes of specific proteins in vivo remains largely uncharacterized, in part due to the challenge of probing protein structures in living cells. Here, we use deep mutational scanning to investigate how a toxic conformation of -synuclein, a dynamic protein linked to Parkinsons disease, responds to perturbations of cellular proteostasis. In the context of a course for graduate students in the UCSF Integrative Program in Quantitative Biology, we screened a comprehensive library of -synuclein missense mutants in yeast cells treated with a variety of small molecules that perturb cellular processes linked to -synuclein biology and pathobiology. We found that the conformation of -synuclein previously shown to drive yeast toxicity--an extended, membrane-bound helix--is largely unaffected by these chemical perturbations, underscoring the importance of this conformational state as a driver of cellular toxicity. On the other hand, the chemical perturbations have a significant effect on the ability of mutations to suppress -synuclein toxicity. Moreover, we find that sequence determinants of -synuclein toxicity are well described by a simple structural model of the membrane-bound helix. This model predicts that -synuclein penetrates the membrane to constant depth across its length but that membrane affinity decreases toward the C terminus, which is consistent with orthogonal biophysical measurements. Finally, we discuss how parallelized chemical genetics experiments can provide a robust framework for inquiry-based graduate coursework.

biochemistry↗