Tight-packing of large pilin subunits provides distinct structural and mechanical properties for the Myxococcus xanthus type IVa pilus.

Type IVa pili (T4aP) are ubiquitous cell surface filaments important for surface motility, adhesion to surfaces, DNA uptake, biofilm formation, and virulence. T4aP are built from thousands of copies of the major pilin subunit and tipped by a complex composed of minor pilins and in some systems also the PilY1 adhesin. While major pilins of structurally characterized T4aP have lengths of <165 residues, the major pilin PilA of Myxococcus xanthus is unusually large with 208 residues. All major pilins have a conserved N-terminal domain and a variable C-terminal domain, and the additional residues of PilA are due to a larger C-terminal domain. We solved the structure of the M. xanthus T4aP (T4aPMx) at a resolution of 3.0 Å using cryo-EM. The T4aPMx follows the structural blueprint of other T4aP with the pilus core comprised of the interacting N-terminal α1-helices, while the globular domains decorate the T4aP surface. The atomic model of PilA built into this map shows that the large C-terminal domain has more extensive intersubunit contacts than major pilins in other T4aP. As expected from these greater contacts, the bending and axial stiffness of the T4aPMx is significantly higher than that of other T4aP and supports T4aP-dependent motility on surfaces of different stiffnesses. Notably, T4aPMx variants with interrupted intersubunit interfaces had decreased bending stiffness, pilus length, and strongly reduced motility. These observations support an evolutionary scenario whereby the large major pilin enables the formation of a rigid T4aP that expands the environmental conditions in which the T4aP system functions.

High-throughput Selection of Human de novo-emerged sORFs with High Folding Potential.

De novo genes emerge from previously noncoding stretches of the genome. Their encoded de novo proteins are generally expected to be similar to random sequences and, accordingly, with no stable tertiary fold and high predicted disorder. However, structural properties of de novo proteins and whether they differ during the stages of emergence and fixation have not been studied in depth and rely heavily on predictions. Here we generated a library of short human putative de novo proteins of varying lengths and ages and sorted the candidates according to their structural compactness and disorder propensity. Using Förster resonance energy transfer combined with Fluorescence-activated cell sorting, we were able to screen the library for most compact protein structures, as well as most elongated and flexible structures. We find that compact de novo proteins are on average slightly shorter and contain lower predicted disorder than less compact ones. The predicted structures for most and least compact de novo proteins correspond to expectations in that they contain more secondary structure content or higher disorder content, respectively. Our experiments indicate that older de novo proteins have higher compactness and structural propensity compared with young ones. We discuss possible evolutionary scenarios and their implications underlying the age-dependencies of compactness and structural content of putative de novo proteins.

Structural dynamics of Na+ and Ca2+ interactions with full-size mammalian NCX.

Cytosolic Ca2+ and Na+ allosterically regulate Na+/Ca2+ exchanger (NCX) proteins to vary the NCX-mediated Ca2+ entry/exit rates in diverse cell types. To resolve the structure-based dynamic mechanisms underlying the ion-dependent allosteric regulation in mammalian NCXs, we analyze the apo, Ca2+, and Na+-bound species of the brain NCX1.4 variant using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and molecular dynamics (MD) simulations. Ca2+ binding to the cytosolic regulatory domains (CBD1 and CBD2) rigidifies the intracellular regulatory loop (5L6) and promotes its interaction with the membrane domains. Either Na+ or Ca2+ stabilizes the intracellular portions of transmembrane helices TM3, TM4, TM9, TM10, and their connecting loops (3L4 and 9L10), thereby exposing previously unappreciated regulatory sites. Ca2+ or Na+ also rigidifies the palmitoylation domain (TMH2), and neighboring TM1/TM6 bundle, thereby uncovering a structural entity for modulating the ion transport rates. The present analysis provides new structure-dynamic clues underlying the regulatory diversity among tissue-specific NCX variants.

Dual-Capped Helical Interface Mimics.

Disruption of protein-protein interactions is medicinally important. Interface helices may be mimicked in helical probes featuring enhanced rigidities, binding to protein targets, stabilities in serum, and cell uptake. This form of mimicry is dominated by stapling between side chains of helical residues: there has been less progress on helical N-caps, and there were no generalizable C-caps. Conversely, in natural proteins, helicities are stabilized and terminated by C- and N-caps but not staples. Bicyclic caps previously introduced by us enable interface helical mimicry featuring rigid synthetic caps at both termini in this work. An unambiguously helical dual-capped system proved to be conformationally stable, binding cyclins A and E, and showed impressive cellular uptake. In addition, the dual-capped mimic was completely resistant to proteolysis in serum over an extended period when compared with "gold standard" hydrocarbon-stapled controls. Dual-capped peptidomimetics are a new, generalizable paradigm for helical interface probe design.

Solution structure and pressure response of thioredoxin-1 of Plasmodium falciparum.

We present here the solution structures of the protein thioredoxin-1 from Plasmodium falciparum (PfTrx-1), in its reduced and oxidized forms. They were determined by high-resolution NMR spectroscopy at 293 K on uniformly 13C-, 15N-enriched, matched samples allowing to identification of even small structural differences. PfTrx-1 shows an α/ß-fold with a mixed five-stranded ß-sheet that is sandwiched between 4 helices in a ß1 α1 ß2 α2 ß3 α3 ß4 ß5 α4 topology. The redox process of the CGPC motif leads to significant structural changes accompanied by larger chemical shift changes from residue Phe25 to Ile36, Thr70 to Thr74, and Leu88 to Asn91. By high-field high-pressure NMR spectroscopy, rare conformational states can be identified that potentially are functionally important and can be used for targeted drug development. We performed these experiments in the pressure range from 0.1 MPa to 200 MPa. The mean combined, random-coil corrected B1* values of reduced and oxidized thioredoxin are quite similar with -0.145 and -0.114 ppm GPa-1, respectively. The mean combined, random-coil corrected B2* values in the reduced and oxidized form are 0.179 and 0.119 ppm GPa-2, respectively. The mean ratios of the pressure coefficients B2/B1 are -0.484 and -0.831 GPa-1 in the reduced and oxidized form respectively. They differ at some points in the structure after the formation of the disulfide bond between C30 and C33. The thermodynamical description of the pressure dependence of chemical shifts requires the assumption of at least three coexisting conformational states of PfTrx-1. These three conformational states were identified in the reduced as well as in the oxidized form of the protein, therefore, they represent sub-states of the two main oxidation states of PfTrx-1.

Performance of SOBA-AD blood test in discriminating Alzheimer's disease patients from cognitively unimpaired controls in two independent cohorts.

Amyloid-beta (Aß) toxic oligomers are critical early players in the molecular pathology of Alzheimer's disease (AD). We have developed a Soluble Oligomer Binding Assay (SOBA-AD) for detection of these Aß oligomers that contain α-sheet secondary structure that discriminates plasma samples from patients on the AD continuum from non-AD controls. We tested 265 plasma samples from two independent cohorts to investigate the performance of SOBA-AD. Testing was performed at two different sites, with different personnel, reagents, and instrumentation. Across two cohorts, SOBA-AD discriminated AD patients from cognitively unimpaired (CU) subjects with 100% sensitivity, > 95% specificity, and > 98% area under the curve (AUC) (95% CI 0.95-1.00). A SOBA-AD positive readout, reflecting α-sheet toxic oligomer burden, was found in AD patients, and not in controls, providing separation of the two populations, aside from 5 SOBA-AD positive controls. Based on an earlier SOBA-AD study, the Aß oligomers detected in these CU subjects may represent preclinical cases of AD. The results presented here support the value of SOBA-AD as a promising blood-based tool for the detection and confirmation of AD.

Hairpin trimer transition state of amyloid fibril.

Protein fibril self-assembly is a universal transition implicated in neurodegenerative diseases. Although fibril structure/growth are well characterized, fibril nucleation is poorly understood. Here, we use a computational-experimental approach to resolve fibril nucleation. We show that monomer hairpin content quantified from molecular dynamics simulations is predictive of experimental fibril formation kinetics across a tau motif mutant library. Hairpin trimers are predicted to be fibril transition states; one hairpin spontaneously converts into the cross-beta conformation, templating subsequent fibril growth. We designed a disulfide-linked dimer mimicking the transition state that catalyzes fibril formation, measured by ThT fluorescence and TEM, of wild-type motif - which does not normally fibrillize. A dimer compatible with extended conformations but not the transition-state fails to nucleate fibril at any concentration. Tau repeat domain simulations show how long-range interactions sequester this motif in a mutation-dependent manner. This work implies that different fibril morphologies could arise from disease-dependent hairpin seeding from different loci.

Structural bases of inhibitory mechanism of CaV1.2 channel inhibitors.

The voltage-gated calcium channel CaV1.2 is essential for cardiac and vessel smooth muscle contractility and brain function. Accumulating evidence demonstrates that malfunctions of CaV1.2 are involved in brain and heart diseases. Pharmacological inhibition of CaV1.2 is therefore of therapeutic value. Here, we report cryo-EM structures of CaV1.2 in the absence or presence of the antirheumatic drug tetrandrine or antihypertensive drug benidipine. Tetrandrine acts as a pore blocker in a pocket composed of S6II, S6III, and S6IV helices and forms extensive hydrophobic interactions with CaV1.2. Our structure elucidates that benidipine is located in the DIII-DIV fenestration site. Its hydrophobic sidechain, phenylpiperidine, is positioned at the exterior of the pore domain and cradled within a hydrophobic pocket formed by S5DIII, S6DIII, and S6DIV helices, providing additional interactions to exert inhibitory effects on both L-type and T-type voltage gated calcium channels. These findings provide the structural foundation for the rational design and optimization of therapeutic inhibitors of voltage-gated calcium channels.

In Vitro Cross-Linking MS Reveals SMG1-UPF2-SMG7 Assembly as Molecular Partners within the NMD Surveillance.

mRNAs containing premature stop codons are responsible for various genetic diseases as well as cancers. The truncated proteins synthesized from these aberrant mRNAs are seldom detected due to the nonsense-mediated mRNA decay (NMD) pathway. Such a surveillance mechanism detects most of these aberrant mRNAs and rapidly destroys them from the pool of mRNAs. Here, we implemented chemical cross-linking mass spectrometry (CLMS) techniques to trace novel biology consisting of protein-protein interactions (PPIs) within the NMD machinery. A set of novel complex networks between UPF2 (Regulator of nonsense transcripts 2), SMG1 (Serine/threonine-protein kinase SMG1), and SMG7 from the NMD pathway were identified, among which UPF2 was found as a connection bridge between SMG1 and SMG7. The UPF2 N-terminal formed most interactions with SMG7, and a set of residues emerged from the MIF4G-I, II, and III domains docked with SMG1 or SMG7. SMG1 mediated interactions with initial residues of UPF2, whereas SMG7 formed very few interactions in this region. Modelled structures highlighted that PPIs for UPF2 and SMG1 emerged from the well-defined secondary structures, whereas SMG7 appeared from the connecting loops. Comparing the influence of cancer-derived mutations over different CLMS sites revealed that variants in the PPIs for UPF2 or SMG1 have significant structural stability effects. Our data highlights the protein-protein interface of the SMG1, UPF2, and SMG7 genes that can be used for potential therapeutic approaches. Blocking the NMD pathway could enhance the production of neoantigens or internal cancer vaccines, which could provide a platform to design potential peptide-based vaccines.

Increased accuracy of vibrational circular dichroism calculations for isotopically labeled helical peptides.

Vibrational circular dichroism (VCD) spectra have been computed with qualitatively correct sign patterns for α-helical peptides using various methods, ranging from empirical models to ab initio quantum mechanical computations. However, some details, such as deuteration effects and isotope substitution shifts and sign patterns for the resultant amide I' band shape, have remained a predictive challenge. Fully optimized computations for a 25-residue Ala-rich peptide, including implicit solvent corrections and explicit side chains that experimentally stabilize these model helical peptides in water, have been carried out using density functional theory (DFT). These fully minimized structures show minor changes in the (ϕ,ψ) torsions at the termini and yield an extra negative band to the low energy side of the characteristic amide I' couplet VCD, in agreement with experiments. Additionally, these calculations give the right sign and relative intensity patterns, as compared to experimental results, for several 13C=O substituted variants. The differences from previously reported computations that used ideal helical structures and vacuum conditions imply that inclusion of distorted termini and solvent effects can have an impact on the final detailed spectral patterns. Inclusion of side chains in these calculations had very little effect on the computed amide I' IR and VCD. Tests of constrained geometries, varying dielectric, and different functionals indicate that each can affect the band shapes, particularly for the 12C=O components, but these aspects do not fully explain the difference from previous spectral simulations. Inclusion of long-range amide coupling, as obtained from DFT computation of the full structure, or transfer of parameters from a somewhat longer peptide model, rather than shorter model, seems to be more important for the final detailed band shape under isotopic substitution. However, these corrections can also induce other changes, suggesting that previously reported, limited calculations may have been qualitatively useful due to a balance of errors. This may also explain the success of simple empirical IR models.

Insights into the dual nature of αB-crystallin chaperone activity from the p.P39L mutant at the N-terminal region.

The substitution of leucine to proline at position 39 (p.P39L) in human αB-crystallin (αB-Cry) has been associated with conflicting interpretations of pathogenicity in cataracts and cardiomyopathy. This study aimed to investigate the effects of the p.P39L mutation on the structural and functional features of human αB-Cry. The mutant protein was expressed in Escherichia coli (E. coli) and purified using anion exchange chromatography. We employed a wide range of spectroscopic analyses, gel electrophoresis, transmission electron microscopy (TEM), and atomic force microscopy (AFM) techniques to investigate the structure, function, stability, and fibrillation propensity of the mutant protein. The p.P39L mutation caused significant changes in the secondary, tertiary, and quaternary structures of human αB-Cry and increased the thermal stability of the protein. The mutant αB-Cry exhibited an increased chaperone activity and an altered oligomeric size distribution, along with an increased propensity to form amyloid aggregates. It is worth mentioning, increased chaperone activity has important positive and negative effects on damaged cells related to cataracts and cardiomyopathy, particularly by interfering in the process of apoptosis. Despite the apparent positive nature of the increased chaperone activity, it is also linked to adverse consequences. This study provides important insights into the effect of proline substitution by leucine at the N-terminal region on the dual nature of chaperone activity in human αB-Cry, which can act as a double-edged sword.

A novel approach for protein secondary structure prediction using encoder-decoder with attention mechanism model.

Computational biology faces many challenges like protein secondary structure prediction (PSS), prediction of solvent accessibility, etc. In this work, we addressed PSS prediction. PSS is based on sequence-structure mapping and interaction among amino acid residues. We proposed an encoder-decoder with an attention mechanism model, which considers the mapping of sequence structure and interaction among residues. The attention mechanism is used to select prominent features from amino acid residues. The proposed model is trained on CB513 and CullPDB open datasets using the Nvidia DGX system. We have tested our proposed method for Q 3 and Q 8 accuracy, segment of overlap, and Mathew correlation coefficient. We achieved 70.63 and 78.93% Q 3 and Q 8 accuracy, respectively, on the CullPDB dataset whereas 79.8 and 77.13% Q 3 and Q 8 accuracy on the CB513 dataset. We observed improvement in SOV up to 80.29 and 91.3% on CullPDB and CB513 datasets. We achieved the results using our proposed model in very few epochs, which is better than the state-of-the-art methods.

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.

Study of Monoclonal Antibody Aggregation at the Air-Liquid Interface under Flow by ATR-FTIR Spectroscopic Imaging.

Throughout bioprocessing, transportation, and storage, therapeutic monoclonal antibodies (mAbs) experience stress conditions that may cause protein unfolding and/or chemical modifications. Such structural changes may lead to the formation of aggregates, which reduce mAb potency and may cause harmful immunogenic responses in patients. Therefore, aggregates need to be detected and removed or ideally prevented from forming. Air-liquid interfaces, which arise during various stages of bioprocessing, are one of the stress factors causing mAb aggregation. In this study, the behavior of an immunoglobulin G (IgG) at the air-liquid interface was investigated under flow using macro attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopic imaging. This chemically specific imaging technique allows observation of adsorption of IgG to the air-liquid interface and detection of associated secondary structural changes. Chemical images revealed that IgG rapidly accumulated around an injected air bubble under flow at 45 °C; however, no such increase was observed at 25 °C. Analysis of the second derivative spectra of IgG at the air-liquid interface revealed changes in the protein secondary structure associated with increased intermolecular ß-sheet content, indicative of aggregated IgG. The addition of 0.01% w/v polysorbate 80 (PS80) reduced the amount of IgG at the air-liquid interface in a static setup at 30 °C; however, this protective effect was lost at 45 °C. These results suggest that the presence of air-liquid interfaces under flow may be detrimental to mAb stability at elevated temperatures and demonstrate the power of ATR-FTIR spectroscopic imaging for studying the structural integrity of mAbs under bioprocessing conditions.

Structural and physical basis for the elasticity of elastin.

Elastin function is to endow vertebrate tissues with elasticity so that they can adapt to local mechanical constraints. The hydrophobicity and insolubility of the mature elastin polymer have hampered studies of its molecular organisation and structure-elasticity relationships. Nevertheless, a growing number of studies from a broad range of disciplines have provided invaluable insights, and several structural models of elastin have been proposed. However, many questions remain regarding how the primary sequence of elastin (and the soluble precursor tropoelastin) governs the molecular structure, its organisation into a polymeric network, and the mechanical properties of the resulting material. The elasticity of elastin is known to be largely entropic in origin, a property that is understood to arise from both its disordered molecular structure and its hydrophobic character. Despite a high degree of hydrophobicity, elastin does not form compact, water-excluding domains and remains highly disordered. However, elastin contains both stable and labile secondary structure elements. Current models of elastin structure and function are drawn from data collected on tropoelastin and on elastin-like peptides (ELPs) but at the tissue level, elasticity is only achieved after polymerisation of the mature elastin. In tissues, the reticulation of tropoelastin chains in water defines the polymer elastin that bears elasticity. Similarly, ELPs require polymerisation to become elastic. There is considerable interest in elastin especially in the biomaterials and cosmetic fields where ELPs are widely used. This review aims to provide an up-to-date survey of/perspective on current knowledge about the interplay between elastin structure, solvation, and entropic elasticity.

Tuning Molecular Motion Enhances Intrinsic Fluorescence in Peptide Amphiphile Nanofibers.

Peptide amphiphiles (PAs) are highly tunable molecules that were recently found to exhibit aggregation-induced emission (AIE) when they self-assemble into nanofibers. Here, we leverage decades of molecular design and self-assembly study of PAs to strategically tune their molecular motion within nanofibers to enhance AIE, making them a highly useful platform for applications such as sensing, bioimaging, or materials property characterization. Since AIE increases when aggregated molecules are rigidly and closely packed, we altered the four most closely packed amino acids nearest to the hydrophobic core by varying the order and composition of glycine, alanine, and valine pairs. Of the six PA designs studied, C16VVAAK2 had the highest quantum yield at 0.17, which is a more than 10-fold increase from other PA designs including the very similar C16AAVVK2, highlighting the importance of precise amino acid placement to anchor rigidity closest to the core. We also altered temperature to increase AIE. C16VVAAK2 exhibited an additional 4-fold increase in maximum fluorescence intensity when the temperature was raised from 5 to 65 °C. As the temperature increased, the secondary structure transitioned from ß-sheet to random coil, indicating that further packing an already aligned molecular system makes it even more readily able to transfer energy between the electron-rich amides. This work both unveils a highly fluorescent AIE PA system design and sheds insights into the molecular orientation and packing design traits that can significantly enhance AIE in self-assembling systems.

Resolving the Nanoscale Structure of ß-Sheet Peptide Self-Assemblies Using Single-Molecule Orientation-Localization Microscopy.

Synthetic peptides that self-assemble into cross-ß fibrils are versatile building blocks for engineered biomaterials due to their modularity and biocompatibility, but their structural and morphological similarities to amyloid species have been a long-standing concern for their translation. Further, their polymorphs are difficult to characterize by using spectroscopic and imaging techniques that rely on ensemble averaging to achieve high resolution. Here, we utilize Nile red (NR), an amyloidophilic fluorogenic probe, and single-molecule orientation-localization microscopy (SMOLM) to characterize fibrils formed by the designed amphipathic enantiomers KFE8L and KFE8D and the pathological amyloid-beta peptide Aß42. Importantly, NR SMOLM reveals the helical (bilayer) ribbon structure of both KFE8 and Aß42 and quantifies the precise tilt of the fibrils' inner and outer backbones in relevant buffer conditions without the need for covalent labeling or sequence mutations. SMOLM also distinguishes polymorphic branched and curved morphologies of KFE8, whose backbones exhibit much more heterogeneity than those of typical straight fibrils. Thus, SMOLM is a powerful tool to interrogate the structural differences and polymorphism between engineered and pathological cross-ß-rich fibrils.

Structural determination and characterisation of the CYP105Q4 cytochrome P450 enzyme from Mycobacterium marinum.

The cytochrome P450 family of heme metalloenzymes (CYPs) catalyse important biological monooxygenation reactions. Mycobacterium marinum contains a gene encoding a CYP105Q4 enzyme of unknown function. Other members of the CYP105 CYP family have key roles in bacterial metabolism including the synthesis of secondary metabolites. We produced and purified the cytochrome P450 enzyme CYP105Q4 to enable its characterization. Several nitrogen-donor atom-containing ligands were found to bind to CYP105Q4 generating type II changes in the UV-vis absorbance spectrum. Based on the UV-vis absorbance spectra none of the potential substrate ligands we tested with CYP105Q4 were able to displace the sixth distal aqua ligand from the heme, though there was evidence for binding of oleic acid and amphotericin B. The crystal structure of CYP105Q4 in the substrate-free form was determined in an open conformation. A computational structural similarity search (Dali) was used to find the most closely related characterized relatives within the CYP105 family. The structure of CYP105Q4 enzyme was compared to the GfsF CYP enzyme from Streptomyces graminofaciens which is involved in the biosynthesis of a macrolide polyketide. This structural comparison to GfsF revealed conformational changes in the helices and loops near the entrance to the substrate access channel. A disordered B/C loop region, usually involved in substrate recognition, was also observed.

Kinetic Ensemble of Tau Protein through the Markov State Model and Deep Learning Analysis.

The ordered assembly of Tau protein into filaments characterizes Alzheimer's and other neurodegenerative diseases, and thus, stabilization of Tau protein is a promising avenue for tauopathies therapy. To dissect the underlying aggregation mechanisms on Tau, we employ a set of molecular simulations and the Markov state model to determine the kinetics of ensemble of K18. K18 is the microtubule-binding domain of Tau protein and plays a vital role in the microtubule assembly, recycling processes, and amyloid fibril formation. Here, we efficiently explore the conformation of K18 with about 150 µs lifetimes in silico. Our results observe that all four repeat regions (R1-R4) are very dynamic, featuring frequent conformational conversion and lacking stable conformations, and the R2 region is more flexible than the R1, R3, and R4 regions. Additionally, it is worth noting that residues 300-310 in R2-R3 and residues 319-336 in R3 tend to form sheet structures, indicating that K18 has a broader functional role than individual repeat monomers. Finally, the simulations combined with Markov state models and deep learning reveal 5 key conformational states along the transition pathway and provide the information on the microsecond time scale interstate transition rates. Overall, this study offers significant insights into the molecular mechanism of Tau pathological aggregation and develops novel strategies for both securing tauopathies and advancing drug discovery.

Unraveling the binding mechanism between soybean protein isolate and selected bioactive compounds.

The present study was aimed to investigate the interactions between soybean protein isolate (SPI) with resveratrol (RESV) and lutein (LUT). The binding forces, molecular interactions and functional properties were explored by multi-spectroscopic analysis, molecular docking and functional property indexes between SPI and RESV/LUT. The RESV/LUT quenched SPI chromophore residues with static mechanism and the endothermic reaction. The SPI- RESV/LUT complexes were formed through hydrogen bond, electrostatic and hydrophobic interactions. Molecular docking confirmed van-der-Waals force as one of the important forces. The interaction of RESV/LUT led to SPI's secondary structure alterations with a decrease in α-helix and random coil and an increase in ß-sheet and ß-turns. RESV/LUT developed foaming and emulsifying properties of SPI and showed a significant decrease of the surface hydrophobicity with RESV/LUT concentrations increase attributed to SPI's partial unfolding. Our study exposed molecular mechanisms and confirmations to understand the interactions in protein- RESV/LUT complexes for protein industrial base promotion.