Protein Conformational Changes Research Articles

Exploring the Activation Process of the Glycine Receptor.

Glycine receptors (GlyR) conduct inhibitory glycinergic neurotransmission in the spinal cord and the brainstem. They play an important role in muscle tone, motor coordination, respiration, and pain perception. However, the mechanism underlying GlyR activation remains unclear. There are five potential glycine binding sites in α1 GlyR, and different binding patterns may cause distinct activation or desensitization behaviors. In this study, we investigated the coupling of protein conformational changes and glycine binding events to elucidate the influence of binding patterns on the activation and desensitization processes of α1 GlyRs. Subsequently, we explored the energetic distinctions between the apical and lateral pathways during α1 GlyR conduction to identify the pivotal factors in the ion conduction pathway preference. Moreover, we predicted the mutational effects of the key residues and verified our predictions using electrophysiological experiments. For the mutants that can be activated by glycine, the predictions of the mutational directions were all correct. The strength of the mutational effects was assessed using Pearson's correlation coefficient, yielding a value of -0.77 between the calculated highest energy barriers and experimental maximum current amplitudes. These findings contribute to our understanding of GlyR activation, identify the key residues of GlyRs, and provide guidance for mechanistic studies on other pLGICs.

Advancing Protein Detection and Analysis Based on Ag/Au PHCN for Enhanced SERS Sensitivity and Specificity in Biomolecular Diagnostics.

In the realm of disease diagnostics, particularly for conditions such as proteinuria and hemoglobinuria, the quest for a method that combines accurate, label-free detection of protein compositions and their conformational changes remains a formidable challenge. In this study, we introduce an innovative Ag/Au plasmonic hybrid coupling nanoarray (Ag/Au PHCN) architecture marked by sub-10 nm interparticle gaps. These nanoarrays, leveraging plasmonic hybrid coupling and synergistic enhancement mechanisms, create a plethora of uniform surface-enhanced Raman spectroscopy (SERS) hotspots. The Ag/Au PHCN substrates demonstrated unparalleled sensitivity in the unmarked detection of hemoglobin (HGB), bovine serum albumin (BSA), and cytochrome C (Cyt.C) in bodily fluids, incorporating the advantages of high sensitivity, high reproducibility, durability, recyclability, and biocompatibility. Notably, the detection limits for BSA and HGB are unprecedented at 0.5 and 5 ng/mL, respectively. This achievement sets a new benchmark for label-free protein detection using two-dimensional nanostructures. Crucially, the Ag/Au PHCNs possess the novel capability to discern protein conformational changes post denaturation, underscoring their potential in probing protein functionalities. Most importantly, these nanoarrays can differentiate between normal and proteinuria-affected urine samples and monitor protein content variations over time, heralding a new era in clinical diagnostics with particular relevance to proteinuria and hemoglobinuria detection.

Atomic-Level Free Energy Landscape Reveals Cooperative Symport Mechanism of Melibiose Transporter.

The Major Facilitator Superfamily (MFS) transporters are an essential class of secondary active transporters involved in various physiological and pathological processes. The melibiose permease (MelB), which catalyzes the stoichiometric symport of the disaccharide melibiose and monovalent cations (e.g., Na+, H+, or Li+), is a key model for understanding the cation-coupled symport mechanisms. Extensive experimental data has established that positive cooperativity between the cargo melibiose and the coupling cation is central to the symport mechanism. However, the structural and energetic origins of this cooperativity remain unclear at the atomistic level for MelB and most other coupled transporters. Here, leveraging recently resolved structures in inward- and outward-facing conformations, we employed the string method and replica-exchange umbrella sampling simulation techniques to comprehensively map the all-atom free energy landscapes of the Na+-coupled melibiose translocation across the MelB in Salmonella enterica serovar Typhimurium (MelBSt), in comparison with the facilitated melibiose transport in a uniporter mutant. The simulation results unravel asymmetrical free energy profiles of melibiose translocation, which is tightly coupled to protein conformational changes in both the N- and C-terminal domains. Notably, the cytoplasmic release of the melibiose induces the simultaneous opening of an inner gate, resulting in a high-energy state of the system. Periplasmic sugar binding and cytoplasmic melibiose released are dynamically coupled with changes in the internal gating elements along the translocation pathway. The outward-facing sugar-bound state is thermodynamically most stable, while the occluded state is a transient state. The binding of Na+ facilitates melibiose translocation by increasing the melibiose-binding affinity and decreasing the overall free energy barrier and change. The cooperative binding of the two substrates results from the allosteric coupling between their binding sites instead of direct electrostatic interaction. These findings add substantial new atomic-level details into how Na+ binding facilitates melibiose translocation and deepen the fundamental understanding of the molecular basis underlying the symport mechanism of cation-coupled transporters.

Enriching Anticancer Drug Pipeline with Potential Inhibitors of Cyclin-Dependent Kinase-8 Identified from Natural Products.

Cyclin-dependent kinase 8 (CDK8) is highly expressed in various cancers and common complex human diseases, and an important therapeutic target for drug discovery and development. The CDK8 inhibitors are actively sought after, especially among natural products. We performed a virtual screening using the ZINC library comprising approximately 90,000 natural compounds. We applied Lipinski's rule of five, absorption, distribution, metabolism, excretion, and toxicity properties, and pan-assay interference compounds filter to eliminate promiscuous binders. Subsequently, the filtered compounds underwent molecular docking to predict their binding affinity and interactions with the CDK8 protein. Interaction analysis were carried out to elucidate the interaction mechanism of the screened hits with binding pockets of the CDK8. The ZINC02152165, ZINC04236005, and ZINC02134595 were selected with appreciable specificity and affinity with CDK8. An all-atom molecular dynamic (MD) simulation followed by essential dynamics was performed for 200 ns. Taken together, the results suggest that ZINC02152165, ZINC04236005, and ZINC02134595 can be harnessed as potential leads in therapeutic development. Moreover, the binding of the molecules brings change in protein conformation in a way that blocks the ATP-binding site of the protein, obstructing its kinase activity. These new findings from natural products offer insights into the molecular mechanisms underlying CDK8 inhibition. CDK8 was previously associated with behavioral and neurological diseases such as autism spectrum disorder, and cancers, for example, colorectal, prostate, breast, and acute myeloid leukemia. Hence, we call for further research and experimental validation, and with an eye to inform future clinical drug discovery and development in these therapeutic fields.

Impedance Spectroscopy Unveiled the Surfactant-Induced Unfolding and Subsequent Refolding of Human Serum Albumin.

Protein-surfactant interaction is a dynamic interplay of electrostatic and hydrophobic forces that ensues from the folding of a protein. We employ impedance spectroscopy (IS), a label-free method, to investigate the unfolding and refolding of human serum albumin (HSA), a globular plasma protein, in the presence of two surfactants: polysorbate-20 (Tween-20), a nonionic surfactant, and sodium dodecyl sulfate (SDS), an anionic surfactant. The equivalent electrical analog circuit was predicted from impedance spectra of HSA in an aqueous solution at physiological pH and room temperature, focusing on varying the concentration of codissolved surfactants. A change in the dielectric constant (ε') and ionic conductivity (κ) is observed by comparing the surfactant-treated protein samples to the bare surfactant solutions to assess the conformational changes induced by surfactants in HSA. Far-UV circular dichroism analysis revealed a decrease in α-helices and an increase in β-sheets and random coils upon SDS addition, which were reversed by Tween-20. Dynamic light scattering supported the findings by measuring changes in the hydrodynamic diameter (dh) of HSA. Unfolding and refolding of HSA with surfactants were also observed through photoluminescence spectroscopy by examining the microenvironment surrounding the single tryptophan (W) within the protein, and the thermodynamic parameters were obtained using the modified Stern-Volmer equation. Our research explores the intriguing domain of protein-surfactant interactions, offering insights with promising applications across diverse biological processes and IS as a suitable alternative technique for investigating protein conformational changes by studying the electrical response of the samples.

Model-free inference of memory in conformational dynamics of a multi-domain protein

Abstract Single-molecule experiments provide insight into the motion (conformational dynamics) of individual protein molecules. Usually, a well-defined but coarse-grained intramolecular coordinate is measured and subsequently analysed with the help of hidden Markov models to deduce the kinetics of protein conformational changes. Such approaches rely on the assumption that the microscopic dynamics of the protein evolve according to a Markov-jump process on some network. However, the manifestation and extent of memory in the dynamics of the observable strongly depends on the chosen underlying Markov model, which is generally not known and therefore can lead to misinterpretations. Here, we combine extensive single-molecule plasmon ruler experiments on the heat shock protein Hsp90, computer simulations, and theory to infer and quantify memory in a model-free fashion. Our analysis is based on the bare definition of non-Markovian behaviour and does not require any underlying model. In the case of Hsp90 probed by a plasmon ruler, the Markov assumption is found to be clearly and conclusively violated on timescales up to roughly 50 s, which corresponds roughly to ∼50% of the inferred correlation time of the signal. The extent of memory is striking and reaches biologically relevant timescales. This implies that memory effects penetrate even the slowest observed motions. We provide clear and reproducible guidelines on how to test for the presence and duration of memory in experimental single-molecule data.

Adaptability to the environment of protease by secondary structure changes and application to enzyme-selective hydrolysis

The reactions involving enzymes are significantly influenced by various environmental factors. Clarity of how the activity and structure of proteases impact their function is crucial for more efficient application of enzymes as a tool. The impact of temperature, pH, and ionic strength on changes in protease activity, secondary structure, and protein conformation during enzymatic hydrolysis were investigated in this study. The enzymatic activity and secondary structure of acid-base protease were found to undergo significant modifications under different physical conditions, as demonstrated by UV spectrophotometry and FTIR spectroscopy analysis. Specifically, variations in α-helix and β-fold content were observed to correlate with changes in enzyme activity. Molecular simulation analysis revealed that physical conditions have varying effects on the protease, particularly influencing enzyme activity and secondary structure. Evaluation of the proteases indicated alterations in both enzyme activity and structure. This treatment selectively hydrolyzed β-lactoglobulin and reduced sensitization. These findings offer novel perspectives on the functionalities and regulatory mechanisms of proteases, as well as potential industrial applications.

Unraveling the mechanism of aroma loss during prolonged hot air drying of non-smoked bacon: Insights into aroma compounds generation and retention

In this study, the potential mechanism of aroma loss in non-smoked bacon due to excessive hot air drying (beyond 24 h) was investigated, focusing on protein conformational changes and the inhibition of heme protein-mediated lipid oxidation by oleic acid. The results showed that prolonged hot-air drying caused a stretching of the myofibrillar protein (MP) conformation in bacon before 36 h, leading to an increase in reactive sulfhydryl groups, surface hydrophobicity, and the exposure of additional hydrophobic sites. Consequently, the binding ability of MP to the eight key aroma compounds (hexanal, 1-octen-3-ol, (E)-2-nonenal, 3-methyl-butanoic acid, 2-undecenal, (E, E)-2,4-decadienal, 2,3-octanedione, and dihydro-5-pentyl-2(3H)-furanone) was enhanced, resulting in their retention. On the other hand, a sustained increase in oleic acid levels has been demonstrated to effectively inhibit heme protein-mediated lipid oxidation and the formation of these key aroma compounds. Using lipidomic techniques, 30 lipid molecules were identified as potential precursors of oleic acid during the bacon drying process. Among these precursors, triglycerides (16:0/18:0/18:1) may be the most significant.

Intermediate Formation via Proton Release during the Photoassembly of the Water-Oxidizing Mn4CaO5 Cluster in Photosystem II.

The early stages of the photoassembly of the water-oxidizing Mn4CaO5 cluster in spinach photosystem II (PSII) were monitored using rapid-scan time-resolved Fourier transform infrared (FTIR) spectroscopy. Carboxylate stretching and the amide I bands, which appeared upon the flash-induced oxidation of a Mn2+ ion, changed their features during the subsequent dark rearrangement process, indicating the relocation of the Mn3+ ion concomitant with protein conformational changes. Monitoring the isotope-edited FTIR signals of a Mes buffer estimated that nearly two protons are released upon the Mn2+ oxidation. Quantum chemical calculations for models of the Mn binding site suggested that the proton of a water ligand is transferred to D1-H332 through a hydrogen bond upon the Mn3+ formation and then released to the bulk as the Mn3+ shifts to bind to this histidine. Another Mn2+ ion may be inserted to form a binuclear Mn3+Mn2+ complex, whose structure was calculated to be stabilized by a μ-hydroxo bridge hydrogen-bonded with deprotonated D1-H337. Nearly one additional proton can thus be released from this histidine, assuming that it is mostly protonated before illumination. Alternatively, a proton could be released by further insertion of Ca2+, forming a Mn3+Mn2+Ca2+ complex with another hydroxo ligand connecting Ca2+ to the Mn3+Mn2+ complex.

Differing temperature dependencies of functional homologs zebrafish Abcb4 and human ABCB1.

The ATP binding cassette (ABC) transporters human ABCB1 and zebrafish (Danio rerio) Abcb4 are functionally homologous multixenobiotic/multidrug (MXR/MDR) efflux transporters that confer the efflux of a broad range of diverse chemical compounds from the cell. As ATPases, the transporters utilize the energy released by ATP cleavage for protein conformation changes and concomitant active transport of substrate compounds. The temperatures, at which human ABCB1 and zebrafish Abcb4 need to function, can substantially differ: Whereas the ambient temperature of human ABCB1, which is that of the human body, is constant, zebrafish Abcb4 has to be active in a wider temperature range as the body temperature of zebrafish can considerably vary, depending on the ambient water temperature (18°C-40°C). Here, we examined the effect of temperature on the ATPase activities of recombinant human ABCB1 and zebrafish Abcb4 generated with the baculovirus expression system. Incubation temperatures for enzyme reactions were set to 37°C and 27°C, corresponding to the human body temperature and the cultivation temperature of zebrafish in our lab, respectively. For stimulation and inhibition of zebrafish Abcb4 and human ABCB1 ATPase activities verapamil and cyclosporin A were added at different concentrations and 50% effect concentrations (EC50) were determined. The different temperatures had a stronger effect on the human ABCB1 than on the zebrafish Abcb4 ATPase: Differences between EC50 values for verapamil at 37°C and 27°C, respectively, were 1.8-fold for human ABCB1 but only 1.2-fold for zebrafish Abcb4. Activation energies (Ea) of basal and verapamil-stimulated ATPases, calculated based on the Arrhenius equation, were 2-fold (basal) and 1.5-fold (verapamil-stimulated) higher for human ABCB1 than for zebrafish Abcb4. The differences between zebrafish Abcb4 and human ABCB1 ATPases in temperature sensitivity and activation energy could be important for the comparison of the functional properties of the two transporter proteins in the context of pharmaco-/toxicokinetics. Related to this, our finding that at equal reaction conditions the zebrafish Abcb4 ATPase activity tended to be generally higher than that of human ABCB1 may also be important, as this may point to a higher substrate compound transport rate of Abcb4.

Beyond the genome: protecting the proteome may be the key to preventing skin aging.

Skin aging is associated with a progressive decline in physiological functions, skin cancers and, ultimately, death. It may be categorized as intrinsic or extrinsic, whereby intrinsic aging is attributed to chronological and genetic factors. At the molecular level, skin aging involves changes in protein conformation and function. The skin proteome changes constantly, mainly through carbonylation; an irreversible phenomenon leading to protein accumulation as toxic aggregates that impair cellular physiology and accelerate skin aging. This review details the central role of proteostasis during skin aging and why proteome protection may be a promising approach in mitigating skin aging. A comprehensive literature review of 87 articles focusing on the proteome, proteostasis, proteotoxicity, protein carbonylation, and the impact of the damaged proteome on aging, and in particular skin aging, was conducted. Skin aging is associated with deficiencies in the repair mechanisms of DNA, transcriptional control, mitochondrial function, cell cycle control, apoptosis, cellular metabolism, changes in hormonal levels secondary to toxicity of damaged proteins, and cell-to-cell communication for tissue homeostasis, which are largely controlled by proteins. In this context, a damaged proteome that leads to the loss of proteostasis may be considered as the first step in tissue aging. There is growing evidence that a healthy proteome plays a central role in skin and in maintaining healthy tissues, thus slowing down the process of skin aging. Hence, protecting the proteome against oxidative or other damage may be an appropriate strategy to prevent and delay skin aging.

A Computational Study of Green Tea Extracts and their Derivatives as Potential Inhibitors for Squalene Monooxygenase.

According to the World Health Organisation, cardiovascular complications have been recognized as the leading course of death between 2000 and 2019. Cardiovascular complications are caused by excess LDL cholesterol in the body or arteries that can build up to form a plaque. There are drugs currently in clinical use called statins that target HMGCoA reductase. However, these drugs result in several side effects. This work investigated using computational approaches to lower cholesterol by investigating green tea extracts as an inhibitors for squalene monooxygenase (the second-rate-controlling step in cholesterol synthesis). Pharmacophore modeling was done to identify possible pharmacophoric sites based on the pIC50 values. The best hypothesis generated by pharmacophore modeling was further validated by atom-based 3D QSAR, where 70% of the data set was treated as the training set. Prior molecular docking ADMET studies were done to investigate the physiochemical properties of these molecules. Glide docking was performed, followed by molecular dynamics to evaluate the protein conformational changes. Pharmacophore results suggest that the best molecules to interact with the biological target should have at least one hydrogen acceptor (A5), two hydrogen donors (D9 and D10), and two benzene rings (R14 and R15) for green tea polyphenols and theasinensin A. ADMET result shows that all molecules in this class have low oral adsorption. Molecular docking results showed that some green tea polyphenols have good binding affinities, with most of these structures having a docking score of less than -10 kcal/mol. Molecular dynamics further illustrated that the best-docked ligands perfectly stay within the active site over a 100 ns simulation. The results obtained from this study suggest that green tea polyphenols have the potential for inhibition of squalene monooxygenase, except for theasinensin A.

Conformation-Dependent Hydrogen-Bonding Interactions in a Switchable Artificial Metalloprotein.

Hydrogen-bonding (H-bonding) interactions in metalloprotein active sites can critically regulate enzyme function. Changes in the protein structure triggered by interplay with substrates, products, and partner proteins are often translated to the metallocofactor by way of specific changes in H-bond networks connected to the active site. However, the complexities of metalloprotein architecture and mechanism often preclude our ability to define the precise molecular interactions giving rise to these intricate regulatory pathways. To address this shortcoming, we have developed conformationally switchable artificial metalloproteins (swArMs) in which allosteric Gln-binding triggers protein conformational changes that impact the microenvironment surrounding an installed metallocofactor. Herein, we report a combined structural, spectroscopic, and computational approach to enhance the conformation-dependent changes in H-bond interactions surrounding the metallocofactor site of a swArM. Structure-informed molecular dynamics simulations were employed to predict point mutations that could enhance active site H-bond interactions preferentially in the Gln-bound holo-conformation of the swArM. Testing our predictions via the unique infrared spectral signals associated with the metallocofactor site, we have identified three key residues capable of imparting conformational control over the metallocofactor microenvironment. The resultant swArMs not only model biologically relevant structural regulation but also provide an enhanced Gln-responsive biological probe to be leveraged in future biosensing applications.

AI-Driven Deep Learning Techniques in Protein Structure Prediction.

Protein structure prediction is important for understanding their function and behavior. This review study presents a comprehensive review of the computational models used in predicting protein structure. It covers the progression from established protein modeling to state-of-the-art artificial intelligence (AI) frameworks. The paper will start with a brief introduction to protein structures, protein modeling, and AI. The section on established protein modeling will discuss homology modeling, ab initio modeling, and threading. The next section is deep learning-based models. It introduces some state-of-the-art AI models, such as AlphaFold (AlphaFold, AlphaFold2, AlphaFold3), RoseTTAFold, ProteinBERT, etc. This section also discusses how AI techniques have been integrated into established frameworks like Swiss-Model, Rosetta, and I-TASSER. The model performance is compared using the rankings of CASP14 (Critical Assessment of Structure Prediction) and CASP15. CASP16 is ongoing, and its results are not included in this review. Continuous Automated Model EvaluatiOn (CAMEO) complements the biennial CASP experiment. Template modeling score (TM-score), global distance test total score (GDT_TS), and Local Distance Difference Test (lDDT) score are discussed too. This paper then acknowledges the ongoing difficulties in predicting protein structure and emphasizes the necessity of additional searches like dynamic protein behavior, conformational changes, and protein-protein interactions. In the application section, this paper introduces some applications in various fields like drug design, industry, education, and novel protein development. In summary, this paper provides a comprehensive overview of the latest advancements in established protein modeling and deep learning-based models for protein structure predictions. It emphasizes the significant advancements achieved by AI and identifies potential areas for further investigation.

Visualizing Single-Molecule Protein Conformational Transitions and Free Energy Landscape.

Monitoring the conformational dynamics of individual proteins is essential to understand the relationship between structure and function in molecular regulatory mechanisms. However, the fast dynamics of single proteins remain poorly understood. Here, we construct a single-molecule sensing platform by introducing plasmonic imaging of single nanoparticles to sense and report the protein conformational changes at the single-molecule level. Tracking the fluctuations of individual nanoparticles with high resolution, we detect and characterize distinct conformational states of molecular chaperone heat shock protein 90 (Hsp90). We also explore the conformational changes of Hsp90 in situ under different nucleotide conditions. Analysis of the conformational fluctuations between the open and closed states of single Hsp90 provides important information on free energy profiles, effective spring constants, and multiphase behaviors. This method offers a strategy to visualize the conformational changes of single proteins in real-time and provides insights into the underlying molecular mechanisms.

Kinetics and mechanisms of thermal deterioration in silver carp (Hypophthalmichtys molitrix) surimi gel quality under high-temperature sterilization.

The gelation properties of surimi gel under various high temperatures (115, 118, and 121 °C) and sterilization intensities (F0 values of 3-7 min) were systematically investigated. A kinetic model detailed quality changes during heat treatment through mathematical analysis, elucidating mechanisms for gel quality degradation. Increased sterilization intensity significantly reduced the quality characteristics of surimi gel. Compared to the gel without sterilization treatment, when the sterilization intensity was increased to 7 min, the gel strength of the groups treated at 115 °C, 118 °C, and 121 °C decreased by 68.35%, 51.4%, and 51.71%, respectively, and the water-holding capacity decreased by 24.87%, 16.85%, and 22.5%, respectively. The hardness, chewiness, and whiteness of the gel also significantly decreased, and the changes in these indicators all conformed to a first-order kinetic model. Activation energy of 291.52 kJ mol-1 highlighted gel strength as the least heat-resistant. At equivalent sterilization intensities, 115 °C exhibited the poorest gel quality, followed by 121 °C, with 118 °C showing relatively better gel quality. Increased T22 and decreased PT22 suggested heightened water mobility and transition of immobilized water within the gel into free water. Protein degradation, weakened disulfide bonds and hydrophobic interaction, and protein conformation changes collectively led to a rough and incoherent gel network structure with large fissures, as verified by the results of scanning electron microscopy. Correlation analysis indicated potential for precise control over surimi gel quality by modulating physicochemical attributes. The outcomes may be beneficial to improve the production and quality control of ready-to-eat surimi-based products. © 2024 Society of Chemical Industry.

Insights into CLC-0's Slow-Gating from Intracellular Proton Inhibition.

The opening of the Torpedo CLC-0 chloride (Cl-) channel is known to be regulated by two gating mechanisms: fast gating and slow (common) gating. The structural basis underlying the fast-gating mechanism is better understood than that of the slow-gating mechanism, which is still largely a mystery. Our previous study on the intracellular proton (H+i)-induced inhibition of the CLC-0 anionic current led to the conclusion that the inhibition results from the slow-gate closure (also called inactivation). The conclusion was made based on substantial evidence such as a large temperature dependence of the H+i inhibition similar to that of the channel inactivation, a resistance to the H+i inhibition in the inactivation-suppressed C212S mutant, and a similar voltage dependence between the current recovery from the H+i inhibition and the recovery from the channel inactivation. In this work, we further examine the mechanism of the H+i inhibition of wild-type CLC-0 and several mutants. We observe that an anion efflux through the pore of CLC-0 accelerates the recovery from the H+i-induced inhibition, a process corresponding to the slow-gate opening. Furthermore, various inactivation-suppressed mutants exhibit different current recovery kinetics, suggesting the existence of multiple inactivated states (namely, slow-gate closed states). We speculate that protonation of the pore of CLC-0 increases the binding affinity of permeant anions in the pore, thereby generating a pore blockage of ion flow as the first step of inactivation. Subsequent complex protein conformational changes further transition the CLC-0 channel to deeper inactivated states.

Revealing the deterioration mechanism in gelling properties of pork myofibrillar protein gel induced by high-temperature treatments: Perspective on the protein aggregation and conformation

The purpose of the present study was to investigate the mechanism of gel deterioration of myofibrillar proteins (MP) gels induced by high-temperature treatments based on the protein aggregation and conformation. The results showed that the gel strength and water holding capacity of MP obviously increased and then decreased as the temperature increased, reaching the maximum value at 80 °C (P < 0.05). The microstructure analysis revealed that appropriate temperature (80 °C) contributed to the formation of a more homogeneous, denser, and smoother three-dimensional mesh structure when compared other treatment temperatures, whereas excessive temperature (95 °C) resulted in the formation of heterogeneous and large protein aggregates of MP, decreasing the continuity of gel networks. This was verified by the rheological properties of MP gels. The particle size (D4,3 and D3,2) of MP obviously increased with larger clusters at excessive temperature, and the surface hydrophobicity of MP decreased (P < 0.05), which has been linked to the formation of soluble or insoluble protein aggregates. Tertiary structure and secondary structure results revealed that the proteins had a tendency to be more stretched under higher temperature treatments, which resulted in a decrease in covalent interactions and non-covalent interactions, fostering the over-aggregation of MP. Therefore, our present study indicated that the degradation of MP gels treated at high temperatures was explained by protein aggregation and conformational changes in MP.

Insight into the Stabilization Mechanism of Succinylation Modification on Black Bean Protein Gels: Molecular Conformation, Microstructure, and Gel Properties.

The objective of this work was to investigate the effect of succinylation treatment on the physicochemical properties of black bean proteins (BBPI), and the relationship mechanism between BBPI structure and gel properties was further analyzed. The results demonstrated that the covalent formation of higher-molecular-weight complexes with BBPI could be achieved by succinic anhydride (SA). With the addition of SA at 10% (v/v), the acylation of proteins amounted to 92.53 ± 1.10%, at which point there was a minimized particle size of the system (300.90 ± 9.57 nm). Meanwhile, the protein structure was stretched with an irregular curl content of 34.30% and the greatest processable flexibility (0.381 ± 0.004). The dense three-dimensional mesh structure of the hydrogel as revealed by scanning electron microscopy was the fundamental prerequisite for the ability to resist external extrusion. The thermally induced hydrogels of acylated proteins with 10% (v/v) addition of SA showed excellent gel elastic behavior (1.44 ± 0.002 nm) and support capacity. Correlation analysis showed that the hydrogel strength and stability of hydrogels were closely related to the changes in protein conformation. This study provides theoretical guidance for the discovery of flexible proteins and their application in hydrogels.

IMAX FRET (Information Maximized FRET) for Multipoint Single-Molecule Structural Analysis.

Understanding the structure of biomolecules is vital for deciphering their roles in biological systems. Single-molecule techniques have emerged as alternatives to conventional ensemble structure analysis methods for uncovering new biology in molecular dynamics and interaction studies, yet only limited structural information could be obtained experimentally. Here, we address this challenge by introducing iMAX FRET, a one-pot method that allows ab initio 3D profiling of individual molecules using two-color FRET measurements. Through the stochastic exchange of fluorescent weak binders, iMAX FRET simultaneously assesses multiple distances on a biomolecule within a few minutes, which can then be used to reconstruct the coordinates of up to four points in each molecule, allowing structure-based inference. We demonstrate the 3D reconstruction of DNA nanostructures, protein quaternary structures, and conformational changes in proteins. With iMAX FRET, we provide a powerful approach to advance the understanding of biomolecular structure by expanding conventional FRET analysis to three dimensions.