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Meat Science Sep 2024This study investigated the effect of ultrasound (US) combined with pre- and post-addition of κ-carrageenan (KC) on the gelling properties, structural characteristics...
Potential mechanisms and effects of ultrasound treatment combined with pre- and post-addition of κ-carrageenan on the gelling properties and rheological behavior of myofibrillar proteins under low-salt condition.
This study investigated the effect of ultrasound (US) combined with pre- and post-addition of κ-carrageenan (KC) on the gelling properties, structural characteristics and rheological behavior of myofibrillar proteins (MP) under low-salt conditions. The results showed that US combined with either pre- or post-addition of KC rendered higher gel strength and water holding capacity (WHC) of MP gels than those treated with US alone and added with KC alone (P < 0.05). US combined with pre-addition of KC facilitated the binding between MP and KC, which enhanced the gel strength and WHC of the mixed MP gels and significantly improved the rheological behavior of MP. This was also confirmed by the highest surface hydrophobicity, disulfide bonds and β-sheet content of the MP gels with US combined with pre-addition of KC. Moreover, microstructural results reflected a denser structure for the pre-addition of KC in combination with US. However, US combined with post-addition of KC resulted in limited MP unfolding and relatively weak hydrophobic interactions in the composite gels, which were less effective in improving the gel properties of the MP gels. This study provides potential strategies for enhancing the gelling properties of low-salt meat products via application of US and KC.
Topics: Carrageenan; Rheology; Animals; Gels; Meat Products; Hydrophobic and Hydrophilic Interactions; Food Handling; Muscle Proteins; Swine; Myofibrils
PubMed: 38838569
DOI: 10.1016/j.meatsci.2024.109554 -
Database : the Journal of Biological... Jun 2024The mechanical stability of proteins is crucial for biological processes. To understand the mechanical functions of proteins, it is important to know the protein...
The mechanical stability of proteins is crucial for biological processes. To understand the mechanical functions of proteins, it is important to know the protein structure and mechanical properties. Protein mechanics is usually investigated through force spectroscopy experiments and simulations that probe the forces required to unfold the protein of interest. While there is a wealth of data in the literature on force spectroscopy experiments and steered molecular dynamics simulations of forced protein unfolding, this information is spread and difficult to access by non-experts. Here, we introduce MechanoProDB, a novel web-based database resource for collecting and mining data obtained from experimental and computational works. MechanoProDB provides a curated repository for a wide range of proteins, including muscle proteins, adhesion molecules and membrane proteins. The database incorporates relevant parameters that provide insights into the mechanical stability of proteins and their conformational stability such as the unfolding forces, energy landscape parameters and contour lengths of unfolding steps. Additionally, it provides intuitive annotations of the unfolding pathways of each protein, allowing users to explore the individual steps during mechanical unfolding. The user-friendly interface of MechanoProDB allows researchers to efficiently navigate, search and download data pertaining to specific protein folds or experimental conditions. Users can visualize protein structures using interactive tools integrated within the database, such as Mol*, and plot available data through integrated plotting tools. To ensure data quality and reliability, we have carefully manually verified and curated the data currently available on MechanoProDB. Furthermore, the database also features an interface that enables users to contribute new data and annotations, promoting community-driven comprehensiveness. The freely available MechanoProDB aims to streamline and accelerate research in the field of mechanobiology and biophysics by offering a unique platform for data sharing and analysis. MechanoProDB is freely available at https://mechanoprodb.ibdm.univ-amu.fr.
Topics: Databases, Protein; Internet; Proteins; User-Computer Interface; Protein Unfolding
PubMed: 38837788
DOI: 10.1093/database/baae047 -
ACS Sensors Jun 2024Hepatic fibrosis, the insidious progression of chronic liver scarring leading to life-threatening cirrhosis and hepatocellular carcinoma, necessitates the urgent...
Hepatic fibrosis, the insidious progression of chronic liver scarring leading to life-threatening cirrhosis and hepatocellular carcinoma, necessitates the urgent development of noninvasive and precise diagnostic methodologies. Denatured collagen emerges as a critical biomarker in the pathogenesis of hepatic fibrosis. Herein, we have for the first time developed 3D-printed collagen capture chips for highly specific surface-enhanced Raman scattering (SERS) detection of denatured type I and type IV collagen in blood, facilitating the early diagnosis of hepatic fibrosis. Employing a novel blend of denatured collagen-targeting peptide-modified silver nanoparticle probes (Ag@DCTP) and polyethylene glycol diacrylate (PEGDA), we engineered a robust ink for the 3D fabrication of these collagen capture chips. The chips are further equipped with specialized SERS peptide probes, Ag@ICTP@R1 (S-I) and Ag@IVCTP@R2 (S-IV), tailored for the targeted detection of type I and IV collagen, respectively. The SERS chip platform demonstrated exceptional specificity and sensitivity in capturing and detecting denatured type I and IV collagen, achieving detection limits of 3.5 ng/mL for type I and 3.2 ng/mL for type IV collagen within a 10-400 ng/mL range. When tested on serum samples from hepatic fibrosis mouse models across a spectrum of fibrosis stages (S0-S4), the chips consistently measured denatured type I collagen and detected a progressive increase in type IV collagen concentration, which correlated with the severity of fibrosis. This novel strategy establishes a benchmark for the multiplexed detection of collagen biomarkers, enhancing our capacity to assess the stages of hepatic fibrosis.
Topics: Liver Cirrhosis; Spectrum Analysis, Raman; Collagen Type I; Animals; Printing, Three-Dimensional; Mice; Collagen Type IV; Silver; Metal Nanoparticles; Protein Denaturation; Humans; Polyethylene Glycols
PubMed: 38836565
DOI: 10.1021/acssensors.4c00623 -
Food & Function Jun 2024The common belief that heat treatment enhances the gastric digestion of proteins is largely based on findings from animal proteins and may not apply to all proteins,...
The common belief that heat treatment enhances the gastric digestion of proteins is largely based on findings from animal proteins and may not apply to all proteins, particularly plant proteins. Here, we compared the digestion characteristics of soybean protein isolates (SPI) in an semi-dynamic digestion model and found distinct effects of heat treatment on the digestion properties of plant proteins. The results revealed that heat-treated SPIs formed clots during the early stages of digestion, although the clots gradually became smaller and looser as digestion progressed, the systems remained turbid at the end of gastric digestion, indicating the lag in their emptying. Furthermore, heat treatment altered the rheological properties of SPI, resulting in increased viscosity and slower gastric emptying. These effects became more pronounced with increasing heat treatment temperatures. The fluorescence spectrum analysis indicated that heat treatment altered its conformation. This led to protein unfolding and exposure of hydrophobic groups, facilitating the formation of larger aggregates during digestion. Additionally, heat treatment exposed more cleavage sites for gastric proteases, increasing the extent of hydrolysis. Elevated levels of free amino acids and a smaller molecular weight distribution further corroborated these findings. These findings contribute to a deeper understanding of the gastric digestion characteristics of plant proteins and the relationship between protein aggregation structure and the digestion process.
Topics: Soybean Proteins; Digestion; Hot Temperature; Protein Aggregates; Glycine max; Humans; Viscosity; Gastric Mucosa; Models, Biological; Rheology; Gastric Emptying
PubMed: 38836315
DOI: 10.1039/d4fo01327a -
ACS Applied Bio Materials Jun 2024Protein compartments offer definitive structures with a large potential design space that are of particular interest for green chemistry and therapeutic applications....
Protein compartments offer definitive structures with a large potential design space that are of particular interest for green chemistry and therapeutic applications. One family of protein compartments, encapsulins, are simple prokaryotic nanocompartments that self-assemble from a single monomer into selectively permeable cages of between 18 and 42 nm. Over the past decade, encapsulins have been developed for a diverse application portfolio utilizing their defined cargo loading mechanisms and repetitive surface display. Although it has been demonstrated that encapsulation of non-native cargo proteins provides protection from protease activity, the thermal effects arising from enclosing cargo within encapsulins remain poorly understood. This study aimed to establish a methodology for loading a reporter protein into thermostable encapsulins to determine the resulting stability change of the cargo. Building on previous reassembly studies, we first investigated the effectiveness of reassembly and cargo-loading of two size classes of encapsulins = 1 and = 3, using superfolder Green Fluorescent Protein. We show that the empty capsid reassembles with higher yield than the capsid and that loading promotes the formation of the = 3 capsid form over the = 1 form, while overloading with cargo results in malformed = 1 encapsulins. For the stability study, a Förster resonance energy transfer (FRET)-probed industrially relevant enzyme cargo, transketolase, was then loaded into the encapsulin. Our results show that site-specific orthogonal FRET labels can reveal changes in thermal unfolding of encapsulated cargo, suggesting that loading of transketolase into the = 1 encapsulin shell increases the thermal stability of the enzyme. This work supports the move toward fully harnessing structural, spatial, and functional control of assembled encapsulins with applications in cargo stabilization.
Topics: Transketolase; Enzyme Stability; Particle Size; Thermotoga maritima; Materials Testing; Biocompatible Materials
PubMed: 38835217
DOI: 10.1021/acsabm.3c01153 -
Journal of the American Society For... Jun 2024Collision-induced unfolding (CIU) of protein ions, monitored by ion mobility-mass spectrometry, can be used to assess the stability of their compact gas-phase fold and...
Collision-induced unfolding (CIU) of protein ions, monitored by ion mobility-mass spectrometry, can be used to assess the stability of their compact gas-phase fold and hence provide structural information. The bacterial elongation factor EF-Tu, a key protein for mRNA translation in prokaryotes and hence a promising antibiotic target, has been studied by CIU. The major [M + 12H] ion of EF-Tu unfolded in collision with Ar atoms between 40 and 50 V, corresponding to an energy of 480-500 eV. Binding of the cofactor analogue GDPNP and the antibiotic enacyloxin IIa stabilized the compact fold of EF-Tu, although dissociation of the latter from the complex diminished its stabilizing effect at higher collision energies. Molecular dynamics simulations of the [M + 12H] EF-Tu ion showed similar qualitative behavior to the experimental results.
PubMed: 38830009
DOI: 10.1021/jasms.4c00087 -
BioRxiv : the Preprint Server For... May 2024Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by the absence of the protein dystrophin. Dystrophin is hypothesized to work as a molecular shock...
Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by the absence of the protein dystrophin. Dystrophin is hypothesized to work as a molecular shock absorber that limits myofiber membrane damage when undergoing reversible unfolding upon muscle stretching and contraction. Utrophin is a dystrophin homologue that is under investigation as a protein replacement therapy for DMD. However, it remains uncertain whether utrophin can mechanically substitute for dystrophin. Here, we compared the mechanical properties of homologous utrophin and dystrophin fragments encoding the N terminus through spectrin repeat 3 (UtrN-R3, DysN-R3) using two operational modes of atomic force microscopy (AFM), constant speed and constant force. Our comprehensive data, including the statistics of force magnitude at which the folded domains unfold in constant speed mode and the time of unfolding statistics in constant force mode, show consistent results. We recover parameters of the energy landscape of the domains and conducted Monte Carlo simulations which corroborate the conclusions drawn from experimental data. Our results confirm that UtrN-R3 expressed in bacteria exhibits significantly lower mechanical stiffness compared to insect UtrN-R3, while the mechanical stiffness of the homologous region of dystrophin (DysN-R3) is intermediate between bacterial and insect UtrN-R3, showing greater similarity to bacterial UtrN-R3.
PubMed: 38826288
DOI: 10.1101/2024.05.18.593686 -
BioRxiv : the Preprint Server For... May 2024Protein-protein complexes can vary in mechanical stability depending on the direction from which force is applied. Here we investigated the anisotropic mechanical...
Protein-protein complexes can vary in mechanical stability depending on the direction from which force is applied. Here we investigated the anisotropic mechanical stability of a molecular complex between a therapeutic non-immunoglobulin scaffold called Affibody and the extracellular domain of the immune checkpoint protein PD-L1. We used a combination of single-molecule AFM force spectroscopy (AFM-SMFS) with bioorthogonal clickable peptide handles, shear stress bead adhesion assays, molecular modeling, and steered molecular dynamics (SMD) simulations to understand the pulling point dependency of mechanostability of the Affibody:(PD-L1) complex. We observed diverse mechanical responses depending on the anchor point. For example, pulling from residue #22 on Affibody generated an intermediate unfolding event attributed to partial unfolding of PD-L1, while pulling from Affibody's N-terminus generated force-activated catch bond behavior. We found that pulling from residue #22 or #47 on Affibody generated the highest rupture forces, with the complex breaking at up to ~ 190 pN under loading rates of ~10-10 pN/sec, representing a ~4-fold increase in mechanostability as compared with low force N-terminal pulling. SMD simulations provided consistent tendencies in rupture forces, and through visualization of force propagation networks provided mechanistic insights. These results demonstrate how mechanostability of therapeutic protein-protein interfaces can be controlled by informed selection of anchor points within molecules, with implications for optimal bioconjugation strategies in drug delivery vehicles.
PubMed: 38826272
DOI: 10.1101/2024.05.21.595133 -
International Journal of Biological... Jun 2024The changes of physicochemical, structural and functional properties and the lysinoalanine (LAL) formation during the unfolding and refolding of black soldier fly larvae...
Changes in physicochemical, structural and functional properties, and lysinoalanine formation during the unfolding and refolding of pH-shifted black soldier fly larvae albumin.
The changes of physicochemical, structural and functional properties and the lysinoalanine (LAL) formation during the unfolding and refolding of black soldier fly larvae albumin (BSFLA) induced by acid/alkaline pH shift were explored. The results showed that acid/alkaline conditions induced unfolding of BSFLA structure, but also accompanied by the formation of some large aggregates due to the hydrophobic interactions, hydrogen bonds, and disulfide bonds. Compared with control or pH shift, pH shift treatment significantly increased the electrostatic repulsion, surface hydrophobicity, free sulfhydryl group, and deamidation reactions, but reduced the fluorescence intensity of BSFLA, and these change in protein conformation contributed to increase in solubility, emulsion activity, and emulsion stability. But the content of LAL in BSFLA was increased by 93.39 % by pH shift treatment. In addition, pH shift modified BSFLA tended to form β-sheet structure through unfolding and refolding, resulting in the formation of aggregates with larger particle sizes, and reducing the solubility and the LAL content by 7.93 % and 65.53 %, respectively. SDS-PAGE profile showed that pH shifting did not cause irreversible denaturation of protein molecules. Therefore, pH-shift is good way to improve the functional properties of BSFLA, but the content of LAL should be reduced to make it better used in food.
Topics: Animals; Hydrogen-Ion Concentration; Protein Unfolding; Lysinoalanine; Hydrophobic and Hydrophilic Interactions; Larva; Albumins; Diptera; Solubility; Protein Refolding; Insect Proteins; Chemical Phenomena
PubMed: 38825263
DOI: 10.1016/j.ijbiomac.2024.132801 -
Biochemical and Biophysical Research... Sep 2024Rab3A is a member of the Rab GTPase family involved in synaptic vesicle trafficking. Recent evidence has demonstrated that Rab3A is phosphorylated by leucine-rich repeat...
Rab3A is a member of the Rab GTPase family involved in synaptic vesicle trafficking. Recent evidence has demonstrated that Rab3A is phosphorylated by leucine-rich repeat kinase 2 (LRRK2) that is implicated in both familial and sporadic forms of Parkinson's disease (PD), and an abnormal increase in Rab3A phosphorylation has been proposed as a cause of PD. Despite the potential importance of Rab3A in PD pathogenesis, its structural information is limited and the effects of bound nucleotides on its biophysical and biochemical properties remain unclear. Here, we show that GDP-bound Rab3A is preferentially phosphorylated by LRRK2 compared with GTP-bound Rab3A. The secondary structure of Rab3A, measured by circular dichroism (CD) spectroscopy, revealed that Rab3A is resistant to heat-induced denaturation at pH 7.4 or 9.0 regardless of the nucleotides bound. In contrast, Rab3A underwent heat-induced denaturation at pH 5.0 at a lower temperature in its GDP-bound form than in its GTP-bound form. The unfolding temperature of Rab3A was studied by differential scanning fluorimetry, which showed a significantly higher unfolding temperature in GTP-bound Rab3A than in GDP-bound Rab3A, with the highest at pH 7.4. These results suggest that Rab3A has unusual thermal stability under physiologically relevant conditions and that bound nucleotides influence both thermal stability and phosphorylation by LRRK2.
Topics: Phosphorylation; Guanosine Triphosphate; Humans; Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; Protein Structure, Secondary; rab3A GTP-Binding Protein; Guanosine Diphosphate; Protein Stability
PubMed: 38824807
DOI: 10.1016/j.bbrc.2024.150199