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Foods (Basel, Switzerland) Aug 2022To investigate the effect of glucose on the protein structure, physicochemical and processing properties of liquid whole eggs (LWE) under homogenization, different...
To investigate the effect of glucose on the protein structure, physicochemical and processing properties of liquid whole eggs (LWE) under homogenization, different concentrations of glucose (0.01, 0.02, 0.04, 0.08 g/mL) were added into LWE, followed by homogenizing at different pressures (5, 10, 20, 40 MPa), respectively. It was shown that the particle size and turbidity of LWE increased with the increase in glucose concentration while decreasing with the increase in homogenization pressure. The protein unfolding was increased at a low concentration of glucose combined with homogenization, indicating a 40.33 ± 5.57% and 165.72 ± 33.57% increase in the fluorescence intensity and surface hydrophobicity under the condition of 0.02 g/mL glucose at 20 MPa, respectively. Moreover, the remarkable increments in foaming capacity, emulsifying capacity, and gel hardness of 47.57 ± 5.1%, 66.79 ± 9.55%, and 52.11 ± 9.83% were recorded under the condition of 0.02 g/mL glucose at 20 MPa, 0.04 g/mL glucose at 20 MPa, and 0.02 g/mL glucose at 40 MPa, respectively. Reasonably, glucose could improve the processing properties of LWE under homogenization, and 0.02 g/mL-0.04 g/mL and 20-40 MPa were the optimal glucose concentration and homogenization pressure. This study could contribute to the production of high-performance and stable quality of LWE.
PubMed: 36010521
DOI: 10.3390/foods11162521 -
Biochemical and Biophysical Research... Jan 2022The thermal shift assay (TSA) is a powerful tool used to detect molecular interactions between proteins and ligands. Using temperature as a physical denaturant and an...
The thermal shift assay (TSA) is a powerful tool used to detect molecular interactions between proteins and ligands. Using temperature as a physical denaturant and an extrinsic fluorescent dye, the TSA tracks protein unfolding. This method precisely determines the midpoint of the unfolding transition (T), which can shift upon the addition of a ligand. Though experimental protocols have been well developed, the thermal shift assay data traditionally yielded qualitative results. Quantitative methods for K determination relied either on empirical and inaccurate usage of T or on isothermal approaches, which do not take full advantage of the melting point precision provided by the TSA. We present a new analysis method based on a model that relies on the equilibrium system between the native and molten globule state of the protein using the van't Hoff equation. We propose the K can be determined by plotting T values versus the logarithm of ligand concentrations and fitting the data to an equation we derived. After testing this procedure with the monomeric maltose-binding protein and an allosterically regulated homotetrameric enzyme (ADP-glucose pyrophosphorylase), we observed that binding results correlated very well with previously established parameters. We demonstrate how this method could potentially offer a broad applicability to a wide range of protein classes and the ability to detect both active and allosteric site binding compounds.
Topics: Adenosine Diphosphate Glucose; Escherichia coli; Glucose-1-Phosphate Adenylyltransferase; Humans; Kinetics; Ligands; Maltose; Maltose-Binding Proteins; Mutagenesis; Protein Unfolding; Proteins; Temperature; Trisaccharides
PubMed: 34959191
DOI: 10.1016/j.bbrc.2021.12.041 -
Biophysical Journal Mar 2020Mechanical processes are involved at many stages of the development of living cells, and often external forces applied to a biomolecule result in its unfolding. Although...
Mechanical processes are involved at many stages of the development of living cells, and often external forces applied to a biomolecule result in its unfolding. Although our knowledge of the unfolding mechanisms and the magnitude of the forces involved has evolved, the role that water molecules play in the mechanical unfolding of biomolecules has not yet been fully elucidated. To this end, we investigated with steered molecular dynamics simulations the mechanical unfolding of dystrophin's spectrin repeat 1 and related the changes in the protein's structure to the ordering of the surrounding water molecules. Our results indicate that upon mechanically induced unfolding of the protein, the solvent molecules become more ordered and increase their average number of hydrogen bonds. In addition, the unfolded structures originating from mechanical pulling expose an increasing amount of the hydrophobic residues to the solvent molecules, and the uncoiled regions adapt a convex surface with a small radius of curvature. As a result, the solvent molecules reorganize around the protein's small protrusions in structurally ordered waters that are characteristic of the so-called "small-molecule regime," which allows water to maintain a high hydrogen bond count at the expense of an increased structural order. We also determined that the response of water to structural changes in the protein is localized to the specific regions of the protein that undergo unfolding. These results indicate that water plays an important role in the mechanically induced unfolding of biomolecules. Our findings may prove relevant to the ever-growing interest in understanding macromolecular crowding in living cells and their effects on protein folding, and suggest that the hydration layer may be exploited as a means for short-range allosteric communication.
Topics: Hydrogen Bonding; Molecular Dynamics Simulation; Protein Folding; Protein Unfolding; Spectrin; Water
PubMed: 32027822
DOI: 10.1016/j.bpj.2020.01.005 -
ACS Omega Jan 2023Mechanoenzymes convert chemical energy from the hydrolysis of nucleotide triphosphates to mechanical energy for carrying out cellular functions ranging from DNA... (Review)
Review
Mechanoenzymes convert chemical energy from the hydrolysis of nucleotide triphosphates to mechanical energy for carrying out cellular functions ranging from DNA unwinding to protein degradation. Protein-processing mechanoenzymes either remodel the protein structures or translocate them across cellular compartments in an energy-dependent manner. Optical-tweezer-based single-molecule force spectroscopy assays have divulged information on details of chemo-mechanical coupling, directed motion, as well as mechanical forces these enzymes are capable of generating. In this review, we introduce the working principles of optical tweezers as a single-molecule force spectroscopy tool and assays developed to decipher the properties such as unfolding kinetics, translocation velocities, and step sizes by protein remodeling mechanoenzymes. We focus on molecular motors involved in protein degradation and disaggregation, i.e., ClpXP, ClpAP, and ClpB, and insights provided by single-molecule assays on kinetics and stepping dynamics during protein unfolding and translocation. Cellular activities such as protein synthesis, folding, and translocation across membranes are also energy dependent, and the recent single-molecule studies decoding the role of mechanical forces on these processes have been discussed.
PubMed: 36643560
DOI: 10.1021/acsomega.2c06044 -
Structure (London, England : 1993) Dec 2021Genetic diversity leads to protein robustness, adaptability, and failure. Some sequence variants are structurally robust but functionally disturbed because mutations...
Genetic diversity leads to protein robustness, adaptability, and failure. Some sequence variants are structurally robust but functionally disturbed because mutations bring the protein onto unfolding/refolding routes resulting in misfolding diseases (e.g., Parkinson). We assume dynamic perturbations introduced by mutations foster the alternative unfolding routes and test this possibility by comparing the unfolding dynamics of the heat-labile enterotoxin B pentamers and the cholera toxin B pentamers, two pentamers structurally and functionally related and robust to 17 sequence variations. The B-subunit thermal unfolding dynamics are monitored by broadband dielectric spectroscopy in nanoconfined and weakly hydrated conditions. Distinct dielectric signals reveal the different B-subunits unfolding dynamics. Combined with network analyses, the experiments pinpoint the role of three mutations A1T, E7D, and E102A, in diverting LTB to alternative unfolding routes that protect LTB from dissociation. Altogether, the methodology diagnoses dynamics faults that may underlie functional disorder, drug resistance, or higher virulence of sequence variants.
Topics: Cholera Toxin; Dielectric Spectroscopy; Enterotoxins; Models, Molecular; Protein Conformation; Protein Folding
PubMed: 34051139
DOI: 10.1016/j.str.2021.05.005 -
Analytical Chemistry Dec 2020Native ion mobility-mass spectrometry (IM-MS) is capable of revealing much that remains unknown within the structural proteome, promising such information on refractory...
Native ion mobility-mass spectrometry (IM-MS) is capable of revealing much that remains unknown within the structural proteome, promising such information on refractory protein targets. Here, we report the development of a unique drift tube IM-MS (DTIM-MS) platform, which combines high-energy source optics for improved collision induced unfolding (CIU) experiments and an electromagnetostatic cell for electron capture dissociation (ECD). We measured a series of high precision collision cross section (CCS) values for protein and protein complex ions ranging from 6-1600 kDa, exhibiting an average relative standard deviation (RSD) of 0.43 ± 0.20%. Furthermore, we compare our CCS results to previously reported DTIM values, finding strong agreement across similarly configured instrumentation (average RSD of 0.82 ± 0.73%), and systematic differences for DTIM CCS values commonly used to calibrate traveling-wave IM separators (-3% average RSD). Our CIU experiments reveal that the modified DTIM-MS instrument described here achieves enhanced levels of ion activation when compared with any previously reported IM-MS platforms, allowing for comprehensive unfolding of large multiprotein complex ions as well as interplatform CIU comparisons. Using our modified DTIM instrument, we studied two protein complexes. The enhanced CIU capabilities enable us to study the gas phase stability of the GroEL 7-mer and 14-mer complexes. Finally, we report CIU-ECD experiments for the alcohol dehydrogenase tetramer, demonstrating improved sequence coverage by combining ECD fragmentation integrated over multiple CIU intermediates. Further improvements for such native top-down sequencing experiments were possible by leveraging IM separation, which enabled us to separate and analyze CID and ECD fragmentation simultaneously.
Topics: Electrons; Mass Spectrometry; Protein Unfolding; Proteins
PubMed: 33166123
DOI: 10.1021/acs.analchem.0c03372 -
Molecular Cell Sep 2022Oxidative stress conditions can cause ATP depletion, oxidative protein unfolding, and potentially toxic protein aggregation. To alleviate this proteotoxic stress, the...
Oxidative stress conditions can cause ATP depletion, oxidative protein unfolding, and potentially toxic protein aggregation. To alleviate this proteotoxic stress, the highly conserved yeast protein, Get3, switches from its guiding function as an ATP-dependent targeting factor for tail-anchored proteins to its guarding function as an ATP-independent molecular chaperone that prevents irreversible protein aggregation. Here, we demonstrate that activation of Get3's chaperone function follows a tightly orchestrated multi-step process, centered around the redox status of two conserved cysteines, whose reactivity is directly controlled by Get3's nucleotide-binding state. Thiol oxidation causes local unfolding and the transition into chaperone-active oligomers. Vice versa, inactivation requires the reduction of Get3's cysteines followed by ATP-binding, which allows the transfer of bound client proteins to ATP-dependent chaperone systems for their effective refolding. Manipulating this fine-tuned cycle of activation and inactivation in yeast impairs oxidative stress resistance and growth, illustrating the necessity to tightly control Get3's intrinsic chaperone function.
Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Guanine Nucleotide Exchange Factors; Molecular Chaperones; Protein Aggregates; Protein Unfolding; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
PubMed: 35839781
DOI: 10.1016/j.molcel.2022.06.015 -
Molecules (Basel, Switzerland) Mar 2022Frataxin (FXN) is a protein involved in storage and delivery of iron in the mitochondria. Single-point mutations in the gene lead to reduced production of functional...
Frataxin (FXN) is a protein involved in storage and delivery of iron in the mitochondria. Single-point mutations in the gene lead to reduced production of functional frataxin, with the consequent dyshomeostasis of iron. FXN variants are at the basis of neurological impairment (the Friedreich's ataxia) and several types of cancer. By using altruistic metadynamics in conjunction with the maximal constrained entropy principle, we estimate the change of free energy in the protein unfolding of frataxin and of some of its pathological mutants. The sampled configurations highlight differences between the wild-type and mutated sequences in the stability of the folded state. In partial agreement with thermodynamic experiments, where most of the analyzed variants are characterized by lower thermal stability compared to wild type, the D104G variant is found with a stability comparable to the wild-type sequence and a lower water-accessible surface area. These observations, obtained with the new approach we propose in our work, point to a functional switch, affected by single-point mutations, of frataxin from iron storage to iron release. The method is suitable to investigate wide structural changes in proteins in general, after a proper tuning of the chosen collective variable used to perform the transition.
Topics: Friedreich Ataxia; Humans; Iron-Binding Proteins; Protein Unfolding; Thermodynamics; Frataxin
PubMed: 35335316
DOI: 10.3390/molecules27061955 -
Proceedings of the National Academy of... Apr 2021Many small proteins move across cellular compartments through narrow pores. In order to thread a protein through a constriction, free energy must be overcome to either...
Many small proteins move across cellular compartments through narrow pores. In order to thread a protein through a constriction, free energy must be overcome to either deform or completely unfold the protein. In principle, the diameter of the pore, along with the effective driving force for unfolding the protein, as well as its barrier to translocation, should be critical factors that govern whether the process proceeds via squeezing, unfolding/threading, or both. To probe this for a well-established protein system, we studied the electric-field-driven translocation behavior of cytochrome (cyt ) through ultrathin silicon nitride (SiN) solid-state nanopores of diameters ranging from 1.5 to 5.5 nm. For a 2.5-nm-diameter pore, we find that, in a threshold electric-field regime of ∼30 to 100 MV/m, cyt is able to squeeze through the pore. As electric fields inside the pore are increased, the unfolded state of cyt is thermodynamically stabilized, facilitating its translocation. In contrast, for 1.5- and 2.0-nm-diameter pores, translocation occurs only by threading of the fully unfolded protein after it transitions through a higher energy unfolding intermediate state at the mouth of the pore. The relative energies between the metastable, intermediate, and unfolded protein states are extracted using a simple thermodynamic model that is dictated by the relatively slow (∼ms) protein translocation times for passing through the nanopore. These experiments map the various modes of protein translocation through a constriction, which opens avenues for exploring protein folding structures, internal contacts, and electric-field-induced deformability.
Topics: Constriction; Cytochromes c; Electricity; Models, Molecular; Nanopores; Protein Folding; Protein Transport; Protein Unfolding; Silicon Compounds; Thermodynamics
PubMed: 33883276
DOI: 10.1073/pnas.2016262118 -
Molecules (Basel, Switzerland) Sep 2019Small GTPases are key regulators of cellular events, and their dysfunction causes many types of cancer. They serve as molecular switches by cycling between inactive... (Review)
Review
Small GTPases are key regulators of cellular events, and their dysfunction causes many types of cancer. They serve as molecular switches by cycling between inactive guanosine diphosphate (GDP)-bound and active guanosine triphosphate (GTP)-bound states. GTPases are deactivated by GTPase-activating proteins (GAPs) and are activated by guanine-nucleotide exchange factors (GEFs). The intrinsic GTP hydrolysis activity of small GTPases is generally low and is accelerated by GAPs. GEFs promote GDP dissociation from small GTPases to allow for GTP binding, which results in a conformational change of two highly flexible segments, called switch I and switch II, that enables binding of the gamma phosphate and allows small GTPases to interact with downstream effectors. For several decades, crystal structures of many GEFs and GAPs have been reported and have shown tremendous structural diversity. In this review, we focus on the latest structural studies of GEFs. Detailed pictures of the variety of GEF mechanisms at atomic resolution can provide insights into new approaches for drug discovery.
Topics: Animals; Enzyme Activation; Feedback, Physiological; Guanine Nucleotide Exchange Factors; Humans; Monomeric GTP-Binding Proteins; Phylogeny; Protein Folding
PubMed: 31514408
DOI: 10.3390/molecules24183308