Maria Stepanova

Genetic variation in cervid PRNP influences chronic wasting disease (CWD) susceptibility and progression, but it remains unclear whether disease-associated substitutions produce localized differences in sequence-derived properties relevant to prion protein (PrPC) condensation. We compared a white-tailed deer (WTD) reference sequence with 10 modeled variants on the same mature PrP scaffold (residues 24–233). For each sequence, we calculated intrinsic disorder, FuzDrop per-residue droplet-promoting propensity (pDP) and binding-mode entropy (Sbind), predicted residue-level pKa values, and a discrete-charge summary at pH 7.0. The overall profiles preserve a long N-terminal droplet-promoting region, whereas variant-associated differences are concentrated in selected intervals. In the FuzDrop outputs, residues 126–140 show allele-dependent changes in pDP near the droplet-promoting threshold, and residues 220–230 show variant-specific changes in Sbind, including lower Sbind for S225F/Y and higher Sbind for elk-like Q226E. These differences occur without large changes in overall intrinsic disorder. KaML pKa predictions identify near-neutral titratable sites that provide electrostatic context for these local predictor differences. Independent atomistic simulations of monomeric PrP further detected variant-sensitive backbone behavior and intramolecular contacts involving these intervals. Together, the analysis identifies residues 126–140 and 220–230 as computationally defined candidate intervals that overlap regions implicated independently in prion conversion or CWD fibril structure, motivating direct tests of cervid PrP condensation and maturation.

We performed a multimodal characterisation of self‐assembled fibrillar aggregates formed by the residue 87–114 fragment of the white‐tailed deer (WTD) prion protein (PrP), a disease‐relevant region implicated in the structural conversion and transmission of chronic wasting disease (CWD) in cervids. Using an integrated experimental platform combining plasmon‐enhanced bright‐field microscopy, label‐free surface‐enhanced Raman spectroscopy (SERS), and Thioflavin T staining, we probed peptide aggregation in solution under ambient conditions. Nanostructured gold films on dielectric fused silica supports served as dual‐purpose plasmonic substrates for imaging and SERS. We observed a transition from dispersed states to well‐defined fibrillar networks, accompanied by spectral evolution in key backbone‐sensitive SERS bands. These results establish the amide III region and tryptophan vibrational features as indicators of fibrillar architecture in SERS assays and offer new insights into the aggregation behavior of amyloidogenic sequences involved in CWD susceptibility.

Prion diseases such as chronic wasting disease involve the conformational conversion of the cellular prion protein (PrP^C) into its misfolded, β-rich isoform (PrP^Sc). While chemical denaturants such as guanidine hydrochloride (GdnHCl) and urea are commonly used to study this process in vitro, their distinct molecular effects on native and misfolded PrP conformers remain incompletely understood. In this study, we employed 500 ns all-atom molecular dynamics simulations and essential collective dynamics analysis to investigate the differential effects of GdnHCl and urea on a composite PrP^C/PrP^Sc system, where white-tailed deer PrP^C interfaces with a corresponding PrP^Sc conformer. GdnHCl was found to preserve interfacial alignment and enhance β-sheet retention in PrP^Sc, while urea promoted partial β-strand dissolution and interfacial destabilization. Both denaturants formed transient contacts with PrP, but urea displaced water hydrogen bonds more extensively. Remarkably, we also observed long-range dynamical coupling across the PrP^C/PrP^Sc interface and between transiently bound solutes and distal protein regions. These findings highlight distinct, denaturant-specific mechanisms of protein destabilization and suggest that localized interactions may propagate non-locally via mechanical or steric pathways. Our results provide molecular-scale insights relevant to prion conversion mechanisms and inform experimental strategies using GdnHCl and urea to modulate misfolding processes in vitro.

Plasmonic nanostructures have received an increasing attention due to their unique ability to mediate conversion of energy of light into different useful forms. This opens pathways for numerous applications from ultrasensitive surface-enhanced Raman scattering (SERS) characterization of materials to heterogeneous photocatalysis and green energy harvesting. However, plasmonic nanostructures should meet a number of requirements for their potential could be realized. In addition to nanoscale dimensions, a high uniformity and compatibility with existing microelectronic settings are required. Electron beam lithography (EBL) offers an unmatched control over nanoscale geometries and also a flexibility to allow for various designs. However, careful co-optimization of EBL exposure and development is required to fabricate periodic patterns with deep nanoscale dimensions. The usage of dielectric substrates is particularly challenging due to the accumulation of charge during EBL exposures. In this work, we have optimized a 10 keV EBL process to fabricate periodic arrays of 50 nm pitch dots on fused silica (FS) supports. To avoid distortions due to charging, a layer of conductive polymer was applied on the surface of the EBL resist, PMMA. In addition, we have investigated the impact of the conductive layer on the PMMA’s exposure by numerical modeling. Despite the predicted significant broadening of the 10 keV electron beam that reaches PMMA after traveling through the conductive layer, quality arrays of dots were successfully fabricated. We used the patterned PMMA as a mask to fabricate 50 nm pitch arrays of Au dots on FS. In order to verify the performance of these Au/FS structures, we used them for SERS biodetection. For this purpose, the samples were biofunctionalized with thiolated DNA aptamers that bind specifically to an important biomarker, protein interleukin 6 (IL-6). The samples were loaded with IL-6 from a solution and characterized by SERS. The results suggest that the fabricated Au/FS plasmonic nanostructures produce an efficient SERS effect. Anticipated multifunctional applications of the plasmonic nanostructures beyond the demonstrated SERS biodetection are discussed.

Alpha-synuclein (α-syn) is a cytoplasmic protein commonly found in the nervous system. In solution α-syn adopts disordered unfolded conformations, although it also can form α-helices upon binding to lipid membranes. Under conditions that are not yet fully understood α-syn can misfold and aggregate giving rise to β-sheet rich amyloid fibrils, which then tend to accumulate in degenerating neurons. This leads to Parkinson's disease (PD) and several other conditions collectively termed synucleinopathies. Development of disease-modifying treatments requires detailed understanding of structure and dynamics of α-syn's misfolded aggregates. We have employed 1000 ns long, all-atom molecular dynamics simulations to investigate the interaction of monomeric α-syn_38-95 fragments, which contain the most important amyloidogenic regions, with pre-formed fibrillar seeds composed of staggered, β-sheet rich α-syn chains of matching length. The simulations indicate that α-syn_38-95 monomers tend to form aggregates with the fibrillar seeds, although we have not observed alignment of the monomeric chains with β-strands of the fibril. To analyze the stability of these aggregates we have employed the essential collective dynamics method, which allows making accurate assessment of dynamical coupling across individual atoms in macromolecules and supramolecular complexes. The analysis revealed extensive dynamical coupling across initially monomeric α-syn chains and the fibrillar seeds including distal regions thereof that did not contact the monomer directly. We have discussed structural origins of these long-range interactions, their impacts for the stability of α-syn aggregates, and potential implications for the development of anti-PD treatments.

Alzheimer’s disease is associated with the formation of toxic aggregates of amyloid beta (Aβ) peptides. Despite tremendous efforts, our understanding of the molecular mechanisms of aggregation, as well as cofactors that might influence it, remains incomplete. The small cyclic neuropeptide somatostatin-14 (SST₁₄) was recently found to be the most selectively enriched protein in human frontal lobe extracts that binds Aβ₄₂aggregates. Furthermore, SST₁₄’s presence was also found to promote the formation of toxic Aβ₄₂oligomersin vitro. In order to elucidate how SST₁₄influences the onset of Aβ oligomerization, we performed all-atom molecular dynamics simulations of model mixtures of Aβ₄₂or Aβ₄₀peptides with SST₁₄molecules and analyzed the structure and dynamics of early-stage aggregates. For comparison we also analyzed the aggregation of Aβ₄₂in the presence of arginine vasopressin (AVP), a different cyclic neuropeptide. We observed the formation of self-assembled aggregates containing the Aβ chains and small cyclic peptides in all mixtures of Aβ₄₂–SST₁₄, Aβ₄₂–AVP, and Aβ₄₀–SST₁₄. The Aβ₄₂–SST₁₄mixtures were found to develop compact, dynamically stable, but small aggregates with the highest exposure of hydrophobic residues to the solvent. Differences in the morphology and dynamics of aggregates that comprise SST₁₄or AVP appear to reflect distinct (1) regions of the Aβ chains they interact with; (2) propensities to engage in hydrogen bonds with Aβ peptides; and (3) solvent exposures of hydrophilic and hydrophobic groups. The presence of SST₁₄was found to impede aggregation in the Aβ₄₂–SST₁₄system despite a high hydrophobicity, producing a stronger “sticky surface” effect in the aggregates at the onset of Aβ₄₂–SST₁₄oligomerization.

We have reported the in situ surface–enhanced Raman spectroscopy (SERS) monitoring of repetitive surface plasmon–mediated chemical transformation cycles in a conjugate nanobiological system. The nanobiological conjugate comprised a gold-coated plasmonic substrate biofunctionalized with thiolated single–stranded DNA carrying a reduction-oxidation indicator methylthioninium chloride, which is also known as methylene blue (MB), in buffer solution at a neutral pH. Exposure to a 523-nm laser excitation produced pronounced SERS bands of oxidized MB. Continued exposure to the laser resulted in disappearance of the SERS bands, which can be interpreted as a reduction of MB. This occurred in the absence of electrochemical stimulation, chemical agents, or catalysts, suggesting a surface plasmon–mediated mechanism of the transformation. The oxidized form of MB was recovered by an addition of fresh buffer solution on the surface of the sample. Continued laser exposure with periodical addition of the buffer resulted in repetitive cycles of changes in the SERS pattern, which were monitored in situ. The chemical transformations of MB were preceded by a buildup of an intermediate SERS pattern, which was attributed to a transient form of MB created by selective surface plasmon-driven excitation.