Biophysical Chemistry

It is important to determine whether native state dynamics drive the misfolding and oligomerization of the prion protein, which are important events in prion disease, and how they are modulated by conformational conversion. Native (N) mouse prion protein (moPrP) is known to form small (OS) and large (OL) oligomers rich in {beta}-sheet, and in this study, photoinduced electron transfer-fluorescence correlation spectroscopy (PET-FCS) has been used to characterize intramolecular dynamics within individual monomeric units in both isolated OS and OL, as well as the diffusion properties of the oligomers. It is estimated that OS and OL comprise of about 15 and 55 monomeric units, respectively. Microsecond dynamics at each of the two regions that are the 1-3 and 2-3 interfaces of native protein are distinct in N, OS and OL, although they occur on very similar timescales. Analysis of the evolution of the distribution of diffusion times, determined using the maximum entropy method, indicates heterogeneity in the oligomerization reaction. Analysis of the change in the fluctuations which occur in two different timescales in the native state ensemble shows that they are damped more at the erstwhile 1-3 interface than the erstwhile 2-3 interface. The difference in the extent of damping at the erstwhile 1-3 and 2-3 interfaces can be explained on the basis of the structural changes known to occur across each region. The changes in dynamics occur concurrently in both regions, indicating that the structural changes accompanying conformational conversion also occur simultaneously during the oligomerization of moPrP.

Amyloid {beta}-protein (A{beta}), which is known for its role in Alzheimer's disease (AD), is also involved in several physiological functions. Of the two predominant isoforms, A{beta}40 and A{beta}42, the latter is more aggregation-prone and more active in health and disease. In the brain, A{beta} interacts with free lipids, which may affect its assembly pathways. Here, thioflavin T (ThT) fluorescence spectroscopy is combined with molecular dynamics (MD) to investigate A{beta}40 and A{beta}42 co-assembly with free dimyristoylphosphatidylcholine (DMPC) and DMPC/cholesterol (CHOL) lipids. ThT measurements at several protein-to-lipid (PL) ratios show isoform- and lipid composition-dependent kinetics of A{beta} fibril formation with clear isoform-dependent lipid effects at a PL ratio of 1:5. Early co-assembly stages of six A{beta}40/A{beta}42 with DMPC and DMPC/CHOL lipids at this PL ratio are then examined by MD. MD results indicate that lipids alter the secondary structure more and interact more avidly with A{beta}40 than with A{beta}42. In turn, A{beta}40 increases the planar order of lipid bicelles more than A{beta}42. Unlike A{beta}40, A{beta}42 decreases the nematic order of CHOL. In DMPC/CHOL lipids, A{beta}42 but not A{beta}40 exhibits two A{beta}-lipid interaction modes associated with mutually exclusive A{beta}42-lipid and A{beta}42-A{beta}42 interactions. Our MD findings provide a plausible explanation for an experimentally observed increase in the lag time for A{beta}40 fibril formation in DMPC lipids and even more so in DMPC/CHOL lipids that contrasts with much smaller effects of lipids on A{beta}42 fibril formation and offer additional atomistic-level insights into A{beta} co-assembly with lipids amenable to experimental testing.

Carbohydrates are essential molecules involved in a wide range of biological processes. While their structural representation in the Protein Data Bank (PDB) has been hindered due to their highly diverse and flexible nature, advancements on X-ray crystallography and CryoEM are accelerating the obtaining of their experimental structures, which in turn supports improved analyses of their structural properties, such as the role of their non-chair conformations on biological processes. In this context, the current work provides a comprehensive and up to date overview of carbohydrate structures within the PDB through classification, quantification, and characterization of monosaccharides and glycan chains, incorporating Cremer-Pople puckering coordinates to evaluate pyranose conformations. Our findings show that, even with a steep increase in CryoEM structures including more complex oligosaccharides, carbohydrate data remains concentrate dinsmallglycanchains, with a about 4% of non-chair conformation sintopresolution structures. Free energy landscapes for the pyranose ring in the presence and absence of nine different binding proteins, including both enzymes and non-enzymes, reinforce the preferred stability of chairs and the rarity of conformational induction upon binding.

Nerve Growth Factor (NGF) supports neuronal survival, plasticity, and growth by binding to p75NTR and TrkA receptors. There is a strong link between NGF and the cholinergic hippocampus, and changes in NGF pathways are observed in Alzheimer's disease (AD). Research shows NGF's neuroprotective and regenerative effects on cholinergic neurons, with positive results in AD models and clinical studies. However, NGF therapy faces challenges: limited blood-brain barrier crossing and dose-dependent pain. To address pain, a mutated NGF called CHF6467 was developed. Thanks to the R100E mutation, it prevents binding to p75NTR while maintaining TrkA affinity, which helps reduce pain side effects. Understanding CHF6467's mechanism remains a challenge. In this context, untargeted metabolomics is effective for uncovering unforeseen responses, making it suitable for assessing CHF6467's overall effects. Building on our previous research and recognizing the importance of amyloid-{beta} peptide as a critical pathological marker of AD, we used NMR-metabolomics to study the effect of CHF6467 on SH-SY5Y cells. The study examined metabolomic profiles of cells treated with CHF6467, with and without A{beta}(1-42), using NMR. Results show CHF6467 influences the metabolic changes caused by A{beta}(1-42), inducing biochemical effects consistent with the promotion of phospholipid precursors, reduced glutamate excitotoxicity, and restored energy metabolism.

Protein aggregates are a diverse array of intra- and extracellular structures containing misfolded proteins with limited macromolecular movement. Protein aggregation is induced by aging and cellular stress and is a hallmark of neurodegenerative diseases including Huntington's, Alzheimer's, and Parkinson's. Fluorescent tools to visualize global protein aggregation in whole organisms are, therefore, important for screening genes and compounds that may reduce or alter protein aggregation in connection with improved health outcomes. Fluorescent probes that report low fluidity structures in cells, such as molecular rotor dyes, are gaining popularity and overcome certain limitations of targeted markers such as tagged proteins or antibodies. However, permeability has been a key barrier preventing widespread use of such probes in whole organisms, including in C. elegans. To achieve visualization of non-specific protein aggregates in whole C. elegans nematodes without the need for strain engineering, we have developed a simple soaking protocol. Our protocol overcomes the permeability limitations of the commercially available molecular rotor dye, Proteostat, enabling widespread labeling of protein aggregates in live and post-fixed worms. Here, we present our novel approach and demonstrate quantification of protein aggregates in multiple tissues to detect differences in protein aggregation due to both genotype and treatment effects. We also outline anticipated future applications for this protocol, including its adaptation for other dyes, as well as limitations and special considerations for its use.

G protein-coupled receptors (GPCRs) represent the largest membrane protein family in the human body and the major targets of FDA-approved medications. Unlike some other physiological proteins, GPCRs are highly plastic and often exist as collections of conformational ensembles in the environment to exert their function. Therefore, understanding how a GPCR adopts different conformational ensembles in response to ligands and signaling partners is essential for drug development. 19F-NMR has emerged as one of the most promising tools in this regard; however, a well-established 19F-labeling system is still lacking, including an effective 19F probe that meets both environmental susceptibility and NMR sensitivity requirements, even though progress has been made dramatically in recent years. Thus, this manuscript reports a novel conformational probe (4-(bromomethyl)-ethoxy-2-(trifluoromethyl)benzene, BTMB) that offers paramount benefits for future GPCR conformational dynamics studies. To rationalize the mechanism behind BTMB's superiority for future probe development, we performed MD simulations, referenced to the current state-of-the-art probe, BTFMA. Our study suggests that the spatial proximity of aromatic residues significantly influences the chemical shift dispersion and linewidths of related 19F-probe-profiled conformational states, as well as the rigidity and compactness of -CF3 in the target protein.

Subtle variations in the activity and signaling of PERK, an endoplasmic reticulum kinase, can shift stress response outcomes. PERK has three commonly occurring exonic single-nucleotide variants frequently inherited together as part of the PERK haplotype B (PERK B), which occurs in 28% of the global population and is associated with several chronic neurodegenerative diseases, although its impact on PERK activity remains controversial. We hypothesized that PERK-B was associated with increased activity, altering stress response and stress tolerance, and that minorly but consistently elevated PERK activity, in combination with additional environmental or genetic risk factors, would lead to adverse outcomes. Comparison of stress response between PERK A, the major PERK haplotype, and PERK B in neuronal cultures using novel knock-in PERK-B mice alongside wild-type PERK A mice revealed differential, transient changes. Following ER stress, astrocyte-containing neuronal cultures, but not neuron-only cultures expressing PERK B exhibited similar levels of eIF2 phosphorylation, the canonical PERK substrate at PERK peak activation point (2h), but showed higher levels at 4 to 6 h, consistent with altered kinetics of PERK signaling. PERK-B neuronal cultures pretreated with a low dose PERK-specific activator exhibited increased vulnerability to N-methyl-D-aspartate-induced death compared to PERK-A neuronal cultures, indicating impaired acquired stress tolerance. Additionally, neuron-astrocyte coculture experiments revealed that the observed difference in acquired stress tolerance was dependent on the astrocyte genotype. The association of PERK-B with transient changes in stress response and reduced stress tolerance with higher vulnerability to cellular stress, albeit transient and small/moderate in size, has clinical relevance based on its potential contribution to the overall genetic vulnerability in neurodegenerative diseases.

Small multifunctional viral membrane proteins called viroporins are attractive drug targets but are difficult to structurally characterize in a lipid-bilayer environment. We have successfully reconstituted the full-length Envelope (E) protein from SARS-CoV-2 in a lipid-bilayer, co-assembled with a native host factor that binds to E, the PDZ domain 2 of ZO1. We have leveraged the enhancement afforded by dynamic nuclear polarization NMR to determine that the ensemble-averaged oligomer state of E is a pentamer. Complexing the protein with a host PDZ domain did not impact the oligomer state, but resulted in structuring of the last four residues of E, and immobilization of the PDZ domain near the lipid surface. Combined with secondary structure information derived from the chemical shifts, these data were used to model an E protein pentamer together with the PDZ protein dimer. Inclusion of a relevant host factor, as well as a complex lipid bilayer that mimics conditions in the ER-Golgi intermediate compartment, approaches native conditions, and addresses the ongoing controversy regarding the functional oligomer state in an infected cell.

Lipid nanoparticles (LNPs) are a leading drug delivery platform for mRNA or other nucleic acid-based therapeutics. LNP platforms are typically composed of ionizable aminolipids, phospholipids, sterols, and lipids tagged with polyethylene glycol. The ionizable aminolipids are typically cationic at low pH, allowing them to encapsulate negatively charged mRNA. The pH at which the ionizable head groups change charge states, the apparent pKa, is crucial to understand the characteristics and formulation of LNP. However, this is difficult to measure as the apparent pKa differs from the pKa of the aminolipids in bulk solution due to the complex chemical and charge environment. In this study, we use molecular dynamics (MD) simulations with alchemical free energy perturbation to calculate the pKa shift as a function of lipid composition, treating LNP-like and vesicle-like environments. Our results show that the pH-dependent structure of the systems affects the trends in pKa shifts in complex ways, driven by factors including surface electrostatics and lipid internalization. Our all-atom simulation protocol allows us to efficiently estimate the apparent pKa of LNP-like models formed with novel ionizable aminolipids and estimate how it will change with composition.

The development of NIR fluorescent dyes with favorable optical properties is of great importance especially for applications within the field of optical imaging. In this study, we report the design, synthesis and photophysical characterization of novel BODIPY-Xanthene hybrid dyes obtained via Knoevenagel condensation between an asymmetrical BODIPY core and the functionalized xanthene aldehydes intermediates. The synthesized dyes ex-hibit absorption (667 nm) and emission bands (720-722 nm) within the NIR region. Detailed UV-Vis and fluo-rescence studies were performed and molar extinction coefficients and stokes shifts also calculated. Moreover, findings highlighting the influence of structural modifications on the optical behavior of the hybrid dyes and their potential utility in optical imaging applications are reported.

Microplastic pollution from homopolymers such as polypropylene (PP), polystyrene (PS), and polyethylene (PE) poses growing health and environmental concerns, highlighting the need for robust polymer-degrading biocatalysts. We previously identified a naturally occurring cysteine-dependent hydrolase (CsdH) from Rhodococcus opacus, featuring a unique Cys160-Lys127-Asp39 catalytic triad and providing a novel mechanism for polyethylene terephthalate (PET) degradation. In this study, we have determined crystal structures of CsdH in complex with PP, PS, PE, and styrene and demonstrated the potential of the enzyme to directly degrade these homopolymers into their respective low molecular weight degradation products using LC-MS. Structural analysis reveals that the enzyme retains a conserved principal access tunnel while undergoing subtle substrate-dependent changes to accommodate different substrates. The enzyme exhibited broad plastic degradation activity with the highest weight loss observed for PE, followed by PP and PS. SEM analysis reveals surface roughness, cracks and grooves, confirming polymer degradation. Further, these results corroborate through FTIR and TGA analysis. These findings establish a strong foundation for enzyme engineering and aim at developing efficient microplastic degradation.

In our past study on human cilia from primary ciliary dyskinesia (PCD) patients using cryo-electron tomography and mass spectrometry, defects in the DNAH5 gene were proven to cause the loss of the entire outer dynein arm along with other proteins. This phenomenon was not observed in the unicellular green alga Chlamydomonas. In this study, we examined the loss of ciliary components caused by defects in various genes, based on proteomic and structural analyses of motile cilia from multiple PCD patients. The absence of single outer arm dynein genes causes a loss or significant decrease in other inner and outer arm dyneins. Interestingly, some patients with outer dynein defects show decreased intraflagellar transport (IFT) proteins, suggesting an influence of cargo components or assembly on transport. These phenomena were not observed in Chlamydomonas, suggesting a more complex mechanism of ciliogenesis in humans despite the high similarity in the final 3D architecture of the 9+2 axoneme. Defects in certain central pair proteins unexpectedly reduced components on the doublets as well. Our results demonstrate protein-protein interactions during stages of human motile ciliogenesis that are more complex than those in unicellular organisms.

Liquid-liquid phase separation (LLPS) of biomacromolecules has been shown to influence a large variety of biological processes via the formation of membraneless organelles. Aberrant LLPS, however, has been implicated in a number of oncological and neurodegenerative disorders. Proteins related to Alzheimer's, Parkinson's diseases and other major amyloidoses have all been shown to form these condensates. Interestingly, some amyloidogenic proteins were demonstrated to form heterotypic droplets, where both molecules coexisted within the same condensate and affected each other's aggregation. While such cross-interactions have been reported numerous times under non-LLPS conditions, the heterotypic droplet assemblies were only shown for a very limited number of amyloidogenic proteins. In this work, we examine heterotypic LLPS of prion proteins and the pro-inflammatory S100A9, whose cross-interaction under non-LLPS conditions we confirmed in a previous work. The results show that this interaction under condensation-promoting conditions enhances their aggregation, leading to the formation of a higher concentration of amyloid structures. These findings suggest that the pro-inflammatory S100A9 may have a critical role in promoting prion protein aggregation via the assembly of heterotypic condensates under high molecular crowding conditions.

Drug targets and vaccines for bacterial diseases are challenging to develop. Computational subtractive genomics and reverse vaccinology offer a viable substitute for conventional drug and vaccine development approaches. In recent years, Pasteurella multocida, a Gram-negative pathogen responsible for severe infections in humans along with major diseases in livestock and poultry, has developed resistance to multiple drugs. As a result, there is an urgent demand for research into reliable, efficient, highly potent, and cost-effective strategies to combat the rise of Pasteurella multocida infections. To address this, the present study integrated an in silico subtractive genomics and reverse vaccinology workflow to pinpoint proteins with potential value as both drug and vaccine targets. From the complete Pasteurella multocida Pm70 proteome, we identified 446 proteins that had no similarities with human proteins, out of which 129 were found to be essential for the pathogens survival. These 129 proteins are therefore potential candidates for drug development. Further metabolic pathway analysis, virulence potential, subcellular localization, and structural analyses revealed two outer membrane proteins, namely, 47 kDa outer membrane protein and lipopolysaccharide-assembly protein LptD, as promising candidates for therapeutic development. Subsequent antigenicity analysis revealed both outer membrane proteins as potential vaccine targets, exhibiting highly favorable antigenicity scores. Candidate B- and T-cell epitopes were systematically mapped from these two targets based on antigenicity, immunogenicity, toxicity, and allergenicity potential and only high-confidence epitopes were assembled using adjuvants and class-specific linkers to construct the final multi-epitope vaccine (MEV). The designed MEV construct was demonstrated to be highly antigenic, non-toxic and non-allergenic to humans, while possessing favorable structural and physicochemical properties, and exhibited strong binding interactions with human toll-like receptors with minimal molecular deformability. Although further experimental validation is recommended to fully assess their efficacy, the present study establishes a solid foundation for the development of novel therapeutics and vaccine candidates against P. multocida, ultimately contributing to the reduction of its rising infections.

Atomistic simulations of intrinsically disordered proteins (IDPs) are notoriously sensitive to force field inaccuracies, either regarding the protein or the water model, yielding inaccurate observables such as compactness, secondary structure propensities, or kinetics. The currently most widely used IDP force fields are Amber99sb-disp (A99disp) and Charmm36m (C36m). A99disp includes a new water model and thus optimized both, the protein and the water interactions. In contrast, C36m used the Tip3p water model and optimized only protein interactions. In many cases, C36m+Tip3p underestimates radii of gyration compared to FRET or SAXS experiments. Such overly compact structural ensembles are believed to arise from an imbalance between protein-protein, protein-water, and water-water interactions, which might be due to Tip3p inaccuracies. Here, we aim at re-balancing these interactions by combining C36m with the Optimal Point Charge (OPC) water model. Recently, this C36m+OPC combination showed improved accuracy for the disordered domain of the measles virus nucleoprotein. Here we present a systematic assessment, comparing C36m+OPC, C36m+Tip3p, C36m+Tip4p, C22*, A03ws, A99sb-ws, and A99disp for five IDPs, as well as a subset of those for five globular proteins, a set of disordered AGQ-repeat peptides, and the fast folding miniprotein CLN025. We compared extensive MD simulations (> 8.5 ms) with SAXS, NMR, circular dichroism, photo-induced electron transfer (PET), T-jump infrared spectroscopy, and X-ray crystallography measurements. We found that combining C36m with OPC improved accuracy over C36m+Tip3p for IDP ensembles without compromising its accuracy for globular proteins. While also the kinetics of the AGQ-peptides were more accurate for C36m+OPC, those of CLN025 folding were less accurate. Overall, C36m+OPC showed similar accuracy as A99sb-ws and A99disp, the latter is currently considered among the most accurate protein force fields.

Structure-aware proteolysis prediction often ranks candidate cleavage sites using solvent accessibility from static protein structures, yet this assumption is rarely tested against native-state cleavage data. We examined this problem using AprX, a serralysin-family protease that readily hydrolyses caseins but shows lower activity toward native whey proteins. Across six bovine milk proteins, sequence compatibility and local scissile-bond accessibility did not separate caseins from whey proteins. In contrast, accessibility fell progressively in whey proteins as the required contiguous substrate window increased, reaching 0% across the working P4-P4' footprint, while all evaluable casein windows remained accessible in isolated-chain ensembles. Local remodelling showed that whey segments could still adopt AprX-compatible backbone geometry. An external thermolysin benchmark rejected a rigid whole-protein clash metric, but did not alter the contiguous-accessibility result. These findings identify footprint-scale accessibility as a distinct structural variable and show why single-residue solvent exposure can overestimate protease accessibility in folded proteins.

Phenylketonuria (PKU) is an autosomal recessive disorder caused by PAH gene defects and can cause irreversible neurological damage as early as the fetal period; however, non-invasive prenatal monitoring methods are lacking. In this study, Pah+/- female mice were mated with Pah+/-; male mice and wild-type male mice, respectively. Urine samples were collected longitudinally from pregnant mice at 10 time points during gestation from Day 1 to Day 19. Data-independent acquisition (DIA) quantitative proteomics combined with GO/KEGG analysis, WGCNA, and a dual-strategy LASSO-random forest approach was used to screen candidate biomarkers. The results showed that stable differentially expressed proteins between groups were detectable as early as Day 1, and the differences persisted throughout gestation and exhibited temporal dynamics. Permutation tests indicated that the probability of random generation of the differential protein combination was low (0.04-0.06), suggesting that it was not randomly generated. The intersection of the two strategies yielded five proteins, GINM1, PRG4, LY6A, NTM, and YIPF3, and after including GLO1, a total of six candidate biomarkers were obtained. The candidate proteins had interaction or co-expression associations with 14 proteins involved in PKU mechanisms, with GLO1 serving as a network hub. This study found that the maternal urine proteome can non-invasively reflect the systemic response induced by fetal-origin PKU, providing proof of concept and candidate biomarker resources for non-invasive early-pregnancy screening of PKU.

Finite-bandgap charge-based FET biosensors are known to be insensitive to electronic tunneling noise in the semiconductor and interference from weakly-charged proteins in physiological samples. However, their signal is still corrupted by pH fluctuations and condensing counterions in the bulk electrolyte. Here, we demonstrate that electrolyte noise is screened by the Stern layer beyond the linear Debye-Huckel limit, when the interfacial potential drop is larger than the thermal voltage. Using a fully nonlinear Gouy-Chapman-Stern (GCS) theory, a universal calibration curve is derived and shown to collapse the low-noise FET signals for miRNA, extracellular vesicles (EV), and protein, captured either directly or through silica and magnetic nanoparticle charge reporters, over eleven decades of analyte concentration. The theory involves the dissociation constant KD and a gain parameter {chi}, which represents the competing effects of the reporter charge and buffer Debye screening that must be large to produce the low-noise GCS condition. The limit of detection (LOD) is shown to be KD/{chi}, which can be optimized by varying the probe density, charge reporter, and buffer solution. An optimized hybridization buffer then yields {chi} ~ 22 (LOD ~1 pM) for miRNA detection, and multivalent capture reduces KD by four orders of magnitude for EV detection (relative to protein detection) with {chi} ~ 34 (LOD ~10 fM).

Golgins are widely described as long coiled-coil proteins that contribute to the structural organisation and trafficking functions of the Golgi apparatus. Although experimental structures have been determined for a limited number of golgin regions, atomic-level information on their extended coiled-coil segments remains scarce, and the oligomeric state, topology, and register of most predicted regions remain unestablished. Here, we characterise a predicted coiled-coil region of the yeast golgin Bug1 (BUG1cc) using structural, biophysical, and computational approaches. X-ray crystallography revealed a parallel, in-register dimeric coiled-coil containing ten heptad repeats and a predominantly hydrophobic core, with specific polar interactions contributing to dimer stabilisation. In solution, BUG1cc was dimeric under SEC-MALS conditions and remained highly alpha-helical across the pH and ionic strength conditions examined. CD measurements revealed pronounced scan-rate-dependent hysteresis, while DSC independently confirmed an asymmetry between heating and cooling transitions. Increasing protein concentration shifted both apparent transition temperatures while preserving thermal hysteresis, supporting chain association and conformational rearrangements in structural recovery. Structure-based simulations indicated that interface contacts and intra-chain helicity are thermodynamically coupled and melt as a single cooperative unit, and that the monomer released on dissociation is compact and only partially helical, so that reassociation proceeds through a coupled folding-binding mechanism whose rate-limiting step is conformational rather than bimolecular. Together, these results establish the molecular architecture of a predicted coiled-coil region of Bug1 and reveal a complex folding landscape in which oligomerisation, secondary-structure recovery, and kinetic barriers are tightly coupled.

The preoptic area (POA) is a key regulator of body temperature, orchestrating behavioral and physiological thermoregulatory functions, including fever, torpor, and homeostatic responses to ambient temperature. Histamine regulates body temperature across animal taxa, including mammals, lower vertebrates, and invertebrates. Hrh1, which encodes the histamine H1 receptor, is expressed in several neuronal populations in the POA with limited overlap with other POA thermoregulatory neurons. Acute activation of POAHrh1 neurons increased body temperature and physical activity. Acute inhibition attenuated the dark phase increase in body temperature without affecting physical activity. There was no effect of inhibition on body temperature during the light phase, fever, cold exposure, or warm exposure. Assessing behavioral thermoregulation, we found that POAHrh1 neurons stimulated nest building in both sexes, possibly contributing to cold-induced nesting behavior. Through projections to the arcuate, dorsomedial hypothalamic and tuberomammillary nuclei, POAHrh1 neurons can increase body temperature and physical activity. Overall, POAHrh1 neurons participate in several thermoregulatory functions, including daily body temperature rhythm regulation, physical activity, and nest building behavior.