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Protein Science : a Publication of the... Jun 2024Wheat germ agglutinin (WGA) demonstrates potential as an oral delivery agent owing to its selective binding to carbohydrates and its capacity to traverse biological...
Wheat germ agglutinin (WGA) demonstrates potential as an oral delivery agent owing to its selective binding to carbohydrates and its capacity to traverse biological membranes. In this study, we employed differential scanning calorimetry and molecular dynamics simulations to comprehensively characterize the thermal unfolding process of both the complete lectin and its four isolated domains. Furthermore, we present the nuclear magnetic resonance structures of three domains that were previously lacking experimental structures in their isolated forms. Our results provide a collective understanding of the energetic and structural factors governing the intricate unfolding mechanism of the complete agglutinin, shedding light on the specific role played by each domain in this process. The analysis revealed negligible interdomain cooperativity, highlighting instead significant coupling between dimer dissociation and the unfolding of the more labile domains. By comparing the dominant interactions, we rationalized the stability differences among the domains. Understanding the structural stability of WGA opens avenues for enhanced drug delivery strategies, underscoring its potential as a promising carrier throughout the gastrointestinal environment.
Topics: Calorimetry, Differential Scanning; Molecular Dynamics Simulation; Nuclear Magnetic Resonance, Biomolecular; Protein Domains; Protein Stability; Wheat Germ Agglutinins
PubMed: 38747397
DOI: 10.1002/pro.5020 -
Research Square Apr 2024The homotetrameric thermosensitive transient receptor potential vanilloid 1-4 (TRPV1-4) channels in sensory neurons are strongly responsive to heat stimuli. However,...
The homotetrameric thermosensitive transient receptor potential vanilloid 1-4 (TRPV1-4) channels in sensory neurons are strongly responsive to heat stimuli. However, their cold activations have not been reported in line with the nonzero heat capacity difference during heat or cold unfolding transitions. Here, along with the experimental examinations of the predicted ring size changes in different domains against the central pore during channel gating at various temperatures, the K169A mutant of reduced human TRPV3 was first found to be activated and inactivated by cold below 42°C. Further thermoring analyses revealed distinct heat and cold unfolding pathways, which resulted in different protein thermostabilities. Thus, both cold and heat unfolding transitions of thermosensitive TRPV1-4 channels may exist once a mutation destabilizes the closed state.
PubMed: 38746116
DOI: 10.21203/rs.3.rs-4285061/v1 -
DNA Repair Jul 2024The ATP-dependent molecular chaperone Cdc48 (in yeast) and its human counterpart p97 (also known as VCP), are essential for a variety of cellular processes, including... (Review)
Review
The ATP-dependent molecular chaperone Cdc48 (in yeast) and its human counterpart p97 (also known as VCP), are essential for a variety of cellular processes, including the removal of DNA-protein crosslinks (DPCs) from the DNA. Growing evidence demonstrates in the last years that Cdc48/p97 is pivotal in targeting ubiquitinated and SUMOylated substrates on chromatin, thereby supporting the DNA damage response. Along with its cofactors, notably Ufd1-Npl4, Cdc48/p97 has emerged as a central player in the unfolding and processing of DPCs. This review introduces the detailed structure, mechanism and cellular functions of Cdc48/p97 with an emphasis on the current knowledge of DNA-protein crosslink repair pathways across several organisms. The review concludes by discussing the potential therapeutic relevance of targeting p97 in DPC repair.
Topics: Valosin Containing Protein; DNA Repair; Humans; Saccharomyces cerevisiae Proteins; DNA; Saccharomyces cerevisiae; DNA Damage; Cell Cycle Proteins; Nuclear Proteins; Adenosine Triphosphatases; DNA-Binding Proteins; Animals; Intracellular Signaling Peptides and Proteins
PubMed: 38744091
DOI: 10.1016/j.dnarep.2024.103691 -
The Journal of Physical Chemistry. B May 2024In-depth characterization of fundamental folding steps of small model peptides is crucial for a better understanding of the folding mechanisms of more complex...
In-depth characterization of fundamental folding steps of small model peptides is crucial for a better understanding of the folding mechanisms of more complex biomacromolecules. We have previously reported on the folding/unfolding kinetics of a model α-helix. Here, we study folding transitions in chignolin (GYDPETGTWG), a short β-hairpin peptide previously used as a model to study conformational changes in β-sheet proteins. Although previously suggested, until now, the role of the Tyr2-Trp9 interaction in the folding mechanism of chignolin was not clear. In the present work, pH-dependent conformational changes of chignolin were characterized by circular dichroism (CD), nuclear magnetic resonance (NMR), ultrafast pH-jump coupled with time-resolved photoacoustic calorimetry (TR-PAC), and molecular dynamics (MD) simulations. Taken together, our results present a comprehensive view of chignolin's folding kinetics upon local pH changes and the role of the Tyr2-Trp9 interaction in the folding process. CD data show that chignolin's β-hairpin formation displays a pH-dependent skew bell-shaped curve, with a maximum close to pH 6, and a large decrease in β-sheet content at alkaline pH. The β-hairpin structure is mainly stabilized by aromatic interactions between Tyr2 and Trp9 and CH-π interactions between Tyr2 and Pro4. Unfolding of chignolin at high pH demonstrates that protonation of Tyr2 is essential for the stability of the β-hairpin. Refolding studies were triggered by laser-induced pH-jumps and detected by TR-PAC. The refolding of chignolin from high pH, mainly due to the protonation of Tyr2, is characterized by a volume expansion (10.4 mL mol), independent of peptide concentration, in the microsecond time range (lifetime of 1.15 μs). At high pH, the presence of the deprotonated hydroxyl (tyrosinate) hinders the formation of the aromatic interaction between Tyr2 and Trp9 resulting in a more disorganized and dynamic tridimensional structure of the peptide. This was also confirmed by comparing MD simulations of chignolin under conditions mimicking neutral and high pH.
Topics: Hydrogen-Ion Concentration; Kinetics; Protein Folding; Molecular Dynamics Simulation; Oligopeptides; Protein Structure, Secondary
PubMed: 38733339
DOI: 10.1021/acs.jpcb.3c08271 -
Foods (Basel, Switzerland) Apr 2024Spirulina protein (SP) is recognized as a nutritious edible microbial protein and holds potential as a natural emulsifier. Due to the inherent challenges SP faces in...
Spirulina protein (SP) is recognized as a nutritious edible microbial protein and holds potential as a natural emulsifier. Due to the inherent challenges SP faces in stabilizing high internal phase emulsions (HIPEs), ultrasonic techniques were utilized for modification. Noticeable alterations in the structural and functional properties of SP were observed following ultrasonic treatment at various power levels (0, 100, 300, and 500 W). Ultrasound treatment disrupted non-covalent interactions within the protein polymer structure, leading to the unfolding of molecular structures and the exposure of hydrophobic groups. Importantly, the particle size of SP was reduced the most at an ultrasonic power of 300 W, and the three-phase contact angle reached its peak at 84.3°. The HIPEs stabilized by SP modified with 300 W ultrasonication have high apparent viscosity and modulus values and strong storage stability under different environmental conditions. Additionally, the encapsulation of curcumin in HIPEs led to improved retention of curcumin across various settings. The bioavailability increased to 35.36, which is 2.8 times higher than the pure oil. These findings suggest that ultrasound-modified SP is a promising emulsifier for HIPEs, and is expected to encapsulate hydrophobic nutrients such as curcumin more effectively.
PubMed: 38731694
DOI: 10.3390/foods13091324 -
Food Science & Nutrition May 2024In contrast to its widespread traditional and popular culinary use to reduce weight, (adzuki beans) was not subjected to sufficient scientific scrutiny. Particularly,...
In contrast to its widespread traditional and popular culinary use to reduce weight, (adzuki beans) was not subjected to sufficient scientific scrutiny. Particularly, its saponins whose role was never investigated before to unveil the beans' antidiabetic and anti-obesity effects. Four vital pancreatic and intestinal carbohydrate enzymes were selected to assess the potency of the triterpenoidal saponins of to bind and activate these proteins through high-precision molecular modeling and dynamics mechanisms with accurate molecular mechanics Generalized Born Surface Area (MMGBSA) energy calculations; thus, recognizing their anti-obesity potential. Our results showed that adzukisaponin VI and adzukisaponin IV were the best compounds in the -amylase and -glucosidase enzymatic grooves, respectively. Adzukisaponin VI and angulasaponin C were the best fitting in the N-termini of sucrase-isomaltose (SI) enzyme, and angulasaponin C was the best scoring compound in maltase-glucoamylase C-termini. All of them outperformed the standard drug acarbose. These compounds in their protein complexes were selected to undergo molecular simulations of the drug-bound protein compared to the apo-protein through 100 ns, which confirmed the consistency of binding to the key amino acid residues in the four enzyme pockets with the least propensity of unfolding. Detailed analysis is given of the different polar and hydrophobic binding interactions of docked compounds. While maltase-adzukisaponin VI complex scored the lowest MMGBSA free energy of -67.77 Kcal/mol, α-amylase complex with angulasaponin B revealed the free binding energy of -74.18 Kcal/mol with a dominance of van der Waals energy (ΔEVDW) and the least change from the start to the end of the simulation time. This study will direct researchers to the significance of isolating the pure adzuki saponin components to conduct future in vitro and in vivo experimental works and even clinical trials.
PubMed: 38726452
DOI: 10.1002/fsn3.4032 -
Protein Science : a Publication of the... Jun 2024One of the most important attributes of anti-amyloid antibodies is their selective binding to oligomeric and amyloid aggregates. However, current methods of examining...
One of the most important attributes of anti-amyloid antibodies is their selective binding to oligomeric and amyloid aggregates. However, current methods of examining the binding specificities of anti-amyloid β (Aβ) antibodies have limited ability to differentiate between complexes that form between antibodies and monomeric or oligomeric Aβ species during the dynamic Aβ aggregation process. Here, we present a high-resolution native ion-mobility mass spectrometry (nIM-MS) method to investigate complexes formed between a variety of Aβ oligomers and three Aβ-specific IgGs, namely two antibodies with relatively high conformational specificity (aducanumab and A34) and one antibody with low conformational specificity (crenezumab). We found that crenezumab primarily binds Aβ monomers, while aducanumab preferentially binds Aβ monomers and dimers and A34 preferentially binds Aβ dimers, trimers, and tetrameters. Through collision induced unfolding (CIU) analysis, our data indicate that antibody stability is increased upon Aβ binding and, surprisingly, this stabilization involves the Fc region. Together, we conclude that nIM-MS and CIU enable the identification of Aβ antibody binding stoichiometries and provide important details regarding antibody binding mechanisms.
Topics: Amyloid beta-Peptides; Antibodies, Monoclonal, Humanized; Ion Mobility Spectrometry; Humans; Mass Spectrometry; Protein Binding; Antibodies, Monoclonal; Protein Multimerization
PubMed: 38723181
DOI: 10.1002/pro.5008 -
Protein Science : a Publication of the... Jun 2024Rieske oxygenases (ROs) are a diverse metalloenzyme class with growing potential in bioconversion and synthetic applications. We postulated that ROs are nonetheless...
Rieske oxygenases (ROs) are a diverse metalloenzyme class with growing potential in bioconversion and synthetic applications. We postulated that ROs are nonetheless underutilized because they are unstable. Terephthalate dioxygenase (TPA PDB ID 7Q05) is a structurally characterized heterohexameric αβ RO that, with its cognate reductase (TPA), catalyzes the first intracellular step of bacterial polyethylene terephthalate plastic bioconversion. Here, we showed that the heterologously expressed TPA/TPA system exhibits only ~300 total turnovers at its optimal pH and temperature. We investigated the thermal stability of the system and the unfolding pathway of TPA through a combination of biochemical and biophysical approaches. The system's activity is thermally limited by a melting temperature (T) of 39.9°C for the monomeric TPA, while the independent T of TPA is 50.8°C. Differential scanning calorimetry revealed a two-step thermal decomposition pathway for TPA with T values of 47.6 and 58.0°C (ΔH = 210 and 509 kcal mol, respectively) for each step. Temperature-dependent small-angle x-ray scattering and dynamic light scattering both detected heat-induced dissociation of TPA subunits at 53.8°C, followed by higher-temperature loss of tertiary structure that coincided with protein aggregation. The computed enthalpies of dissociation for the monomer interfaces were most congruent with a decomposition pathway initiated by β-β interface dissociation, a pattern predicted to be widespread in ROs. As a strategy for enhancing TPA stability, we propose prioritizing the re-engineering of the β subunit interfaces, with subsequent targeted improvements of the subunits.
Topics: Enzyme Stability; Oxidoreductases; Bacterial Proteins; Models, Molecular; Dioxygenases; Temperature; Escherichia coli; Polyethylene Terephthalates; Hydrogen-Ion Concentration; Electron Transport Complex III
PubMed: 38723110
DOI: 10.1002/pro.4997 -
Journal of Food Science Jun 2024This study investigated preheated (25-100°C) black soybean protein isolate (BSPI) conjugated with syringic acid (SA) (25 and 50 µmol/g protein) under alkaline...
This study investigated preheated (25-100°C) black soybean protein isolate (BSPI) conjugated with syringic acid (SA) (25 and 50 µmol/g protein) under alkaline conditions, focusing on the structure, functional properties, and storage stability. The results revealed that the SA binding equivalent and binding rate on BSPI increased continuously as the preheat temperature increased. Additionally, preheating positively impacted the surface hydrophobicity (H) of BSPI, with further enhancement observed upon SA binding. Preheating and SA binding altered the secondary and tertiary structure of BSPI, resulting in protein unfolding and increased molecular flexibility. The improvement in BSPI functional properties was closely associated with both preheating temperature and SA binding. Specifically, preheating decreased the solubility of BSPI but enhanced the emulsifying activity index (EAI) and foaming capacity (FC) of BSPI. Conversely, SA binding increased the solubility of BSPI with an accompanying increase in EAI, FC, foaming stability, and antioxidant activity. Notably, the BSPI100-SA50 exhibited the most significant improvement in functional properties, particularly in solubility, emulsifying, and foaming attributes. Moreover, the BSPI-SA conjugates demonstrated good stability of SA during storage, which positively correlated with the preheating temperature. This study proposes a novel BSPI-SA conjugate with enhanced essential functional properties, underscoring the potential of preheated BSPI-SA conjugates to improve SA storage stability. PRACTICAL APPLICATION: Preheated BSPI-SA conjugates can be used as functional ingredients in food or health products. In addition, preheated BSPI shows potential as a candidate for encapsulating and delivering hydrophobic bioactive compounds.
Topics: Soybean Proteins; Hydrophobic and Hydrophilic Interactions; Solubility; Hot Temperature; Gallic Acid; Glycine max; Antioxidants; Protein Stability
PubMed: 38720591
DOI: 10.1111/1750-3841.17087 -
Quarterly Reviews of Biophysics May 2024Molecular motors are machines essential for life since they convert chemical energy into mechanical work. However, the precise mechanism by which nucleotide binding,... (Review)
Review
Molecular motors are machines essential for life since they convert chemical energy into mechanical work. However, the precise mechanism by which nucleotide binding, catalysis, or release of products is coupled to the work performed by the molecular motor is still not entirely clear. This is due, in part, to a lack of understanding of the role of force in the mechanical-structural processes involved in enzyme catalysis. From a mechanical perspective, one promising hypothesis is the Haldane-Pauling hypothesis which considers the idea that part of the enzymatic catalysis is strain-induced. It suggests that enzymes cannot be efficient catalysts if they are fully complementary to the substrates. Instead, they must exert strain on the substrate upon binding, using enzyme-substrate energy interaction (binding energy) to accelerate the reaction rate. A novel idea suggests that during catalysis, significant strain energy is built up, which is then released by a local unfolding/refolding event known as 'cracking'. Recent evidence has also shown that in catalytic reactions involving conformational changes, part of the heat released results in a center-of-mass acceleration of the enzyme, raising the possibility that the heat released by the reaction itself could affect the enzyme's integrity. Thus, it has been suggested that this released heat could promote or be linked to the cracking seen in proteins such as adenylate kinase (AK). We propose that the energy released as a consequence of ligand binding/catalysis is associated with the local unfolding/refolding events (cracking), and that this energy is capable of driving the mechanical work.
Topics: Animals; Humans; Molecular Motor Proteins; Protein Unfolding; Enzymes; Energy Metabolism
PubMed: 38715547
DOI: 10.1017/S0033583524000052