Biology subjects

Swetman, W. S.

Publications and source records attributed to Swetman, W. S..

4 recordsLinked to original sources

bioRxiv2026

Disulfide Crosslink Density is a Key Determinant in the Formation, Dynamics, and Maturation of Cysteine-rich Designer Biomolecular Condensates.

Biomolecular condensates form through weak, multivalent, non-covalent interactions, but the role of covalent crosslinks in shaping their assembly and material properties remains poorly understood. We previously showed that disulfide bonds between cysteines in designer phase-separating peptides (PSPs) act as redox-reversible crosslinks that lower the saturation concentration (Csat) and help maintain condensate fluidity. Here, we ask which sequence features govern this behavior. We built two peptide libraries: one varying the inter-cysteine spacer length (2-10 residues) and one varying cysteine density (0-33%). Inter-cysteine spacers tuned condensate properties only modestly. Longer inter-cysteine lengths raised Csat from 300 to 900 M, showed greater fluidity (as assessed by fluorescence recovery after photobleaching (FRAP) recovery rate, and resisted loss of fluidity during maturation; all inter-cysteine length variants dissolved and reformed reversibly over three redox cycles. Cysteine density, in contrast, was the dominant control parameter. Increasing cysteine density lowered Csat about 60-fold (1.5 mM to 25 M) and reduced the fluidity by an order of magnitude. Its effect on fluidity was non-monotonic: condensates with up to ~14% cysteine density stayed liquid-like for 10 days, whereas those with 21-33% cysteine density in the peptide showed slower coalescence. Condensates with the highest densities also resisted dissolution under reducing conditions and showed sharply reduced uptake of a protein cargo (eGFP; ~60% to ~9%) and a small molecule (TMR, ~70% to ~45%), consistent with smaller mesh size. Together, these results identify disulfide crosslink density as a key lever for programming condensate assembly, viscoelasticity, redox responsiveness, and porosity. The results offer key design principles both for understanding cysteine-rich disordered proteins in biomolecular condensates and for engineering redox-responsive biomaterials.

biophysics↗

bioRxiv2026

Leveraging Supramolecular Polymers to Induce the Targeted Protein Degradation of α-Synuclein

Halting the progression of neurodegenerative diseases remains one of the foremost challenges in medicinal chemistry due to the complex biology that drives disease progression. For example, a hallmark of synucleinopathies, such as Parkinsons disease, is the misfolding and aggregation of the protein -Synuclein (-Syn), driving the formation of toxic oligomers and fibrils that avoid natural intracellular clearance mechanisms, participate in unusual protein-protein interactions, and ultimately contribute to the death of dopaminergic neurons. The field of targeted protein degradation (TPD) has emerged as an innovative therapeutic route to selectively degrade proteins of interest that leverage natural intracellular protein degradation machinery. First generation TPD therapeutics have traditionally been designed as bifunctional, chimeric compounds in which a short covalent linker tethers a ligand designed to bind target proteins to a ligand that initiates an either proteosome- or lysosome-dependent protein degradation cascade. While initial studies have indicated the promise of these approaches, translation to the clinical setting has been challenging due to difficulties in achieving cellular internalization, long-term stability, and establishment of a generalizable strategy. To overcome these obstacles, this work has focused on adding modularity and dynamic capability to this classical model by leveraging a multivalent macromolecular approach to TPD. Specifically, peptide amphiphiles (PAs) were designed to self-assemble into high-aspect-ratio supramolecular nanofibers and present peptide epitopes on the surface of the fibers to target simultaneous binding of -Syn and recruitment of enzymes that facilitate entry into the lysosome-dependent chaperone-mediated autophagy protein degradation pathway. In vitro application of these bioactive PA nanofibers has demonstrated the ability to independently internalize in cells and reduce -Syn protein levels selectively and effectively. While further optimization of this model has the potential to be a viable therapeutic against -Syn aggregation, the modularity of these supramolecular nanofibers through facile monomer design and incorporation illustrates the potential of establishing a platform technology for targeting a diverse range of pathologic proteins. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=141 SRC="FIGDIR/small/739556v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@d031adorg.highwire.dtl.DTLVardef@6d792dorg.highwire.dtl.DTLVardef@12e80c1org.highwire.dtl.DTLVardef@71a2e_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2025

Disulfide cross-linked redox-sensitive peptide condensates are efficient cell delivery vehicles of molecular cargo.

Biomolecular condensates (BCs) are phase-separated viscoelastic hubs within demixed solutions enriched in proteins and nucleic acids. Such condensates, also called membraneless organelles, are increasingly observed in cells and serve as transient hubs for spatial organization and compartmentalization of biomolecules. Along with the transiency of formation and dissolution, their ability to sequester molecules has inspired us to develop BCs as potential vehicles to transport and deliver molecular cargo. We recently reported the design of disulfide bond cross-linked phase-separating peptide (PSP) condensates that spontaneously dissolve in reducing conditions (JACS, 2024, 146, 255299). Based on the premise that the highly reducing cytoplasm could dissolve PSP condensates and release partitioned cargo, here, we demonstrate the ability of PSP condensates to deliver molecular cargo to the cytoplasm of HeLa cells efficiently. We show that PSP condensates deliver a variety of cargos that differ in their sizes and chemistries, including small molecules, peptides, GFP protein (31 kDa), DNA (1.7 kbp), and mRNA. The transfection efficiencies of PSP condensates for delivering DNA and mRNA were also significantly greater than those of a commercial transfection agent. With room to tailor the condensate properties based on cargo and cell types, these results showcase the potential of disulfide-cross-linked PSPs as effective and customizable cellular delivery vehicles, filling a critical demand gap for such delivery systems.

biophysics↗

bioRxiv2024

Reversible disulfide bond crosslinks as tunable levers of phase separation in designer biomolecular condensates.

Biomolecular condensates (BCs) are membraneless hubs enriched in proteins and nucleic acids that have become important players in many cellular functions. Uncovering the sequence determinants of proteins for phase separation is important in understanding the biophysical and biochemical properties of BCs. Despite significant discoveries in the last decade, the role of cysteine residues in BC formation and dissolution has remained unknown. Here, to determine the involvement of disulfide crosslinks and their redox sensitivity in BCs, we designed a stickers and spacers model of phase-separating peptides interspersed with cysteines. Through biophysical investigations, we learned that cysteines promote liquid-liquid phase separation in oxidizing conditions and perpetuate liquid condensates through disulfide crosslinks, which can be reversibly tuned with redox chemistry. By varying the composition of cysteines, subtle but distinct changes in the viscoelastic behavior of the condensates were observed. Empirically, we conclude that cysteines are neither stickers nor spacers but function as covalent nodes to lower the effective concentrations for sticker interactions and inhibit system-spanning percolation networks. Together, we unmask the role of cysteines in protein phase behavior and the potential to develop tunable, redox-sensitive viscoelastic materials.

biophysics↗