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Progress in Biophysics and Molecular... Jan 2020One of the important physicochemical features of the proteins specifically multi-subunit types is their stability at high temperatures. The kinetics of the dissociation... (Review)
Review
One of the important physicochemical features of the proteins specifically multi-subunit types is their stability at high temperatures. The kinetics of the dissociation and denaturation of proteins possessing at least two subunits has certain challenges because the overall mechanism of dissociation can include hidden reversible and/or irreversible steps (conformational lock). There are numerous proteins related to diseases which are in equilibrium with oligomer forms. This equilibrium plays an important role in holding the activity levels of these proteins in vitro and in vivo. The stability of proteins is an essential milestone to study conformational changes of the proteins in the living cell. This concept is of great importance when proteins are used in biomedicine fields. The quaternary structure of multi-subunit proteins includes properties that affect function and kinetics of denaturation. The kinetics of denaturation may include some hidden stages of conformational transitions in the protein and steps of reversible dissociation of the oligomer. The transforming factors affect different areas especially active centers in the clefts of each subunit. The distinctive agent among all, is the temperature which sequentially destructs independent inter-subunit contact sites. Recent experimental data regarding dissociative mechanism for irreversible thermal denaturation of multi-subunit proteins have been summarized in the present paper.
Topics: Enzymes; Hot Temperature; Kinetics; Models, Molecular; Phase Transition; Protein Conformation; Protein Denaturation; Protein Stability; Proteins; Thermodynamics
PubMed: 31470027
DOI: 10.1016/j.pbiomolbio.2019.08.008 -
International Journal of Molecular... Mar 2023We review the key steps leading to an improved analysis of thermal protein unfolding. Thermal unfolding is a dynamic cooperative process with many short-lived... (Review)
Review
We review the key steps leading to an improved analysis of thermal protein unfolding. Thermal unfolding is a dynamic cooperative process with many short-lived intermediates. Protein unfolding has been measured by various spectroscopic techniques that reveal structural changes, and by differential scanning calorimetry (DSC) that provides the heat capacity change C(T). The corresponding temperature profiles of enthalpy ΔH(T), entropy ΔS(T), and free energy ΔG(T) have thus far been evaluated using a chemical equilibrium two-state model. Taking a different approach, we demonstrated that the temperature profiles of enthalpy ΔH(T), entropy ΔS(T), and free energy ΔG(T) can be obtained directly by a numerical integration of the heat capacity profile C(T). DSC thus offers the unique possibility to assess these parameters without resorting to a model. These experimental parameters now allow us to examine the predictions of different unfolding models. The standard two-state model fits the experimental heat capacity peak quite well. However, neither the enthalpy nor entropy profiles (predicted to be almost linear) are congruent with the measured sigmoidal temperature profiles, nor is the parabolic free energy profile congruent with the experimentally observed trapezoidal temperature profile. We introduce three new models, an empirical two-state model, a statistical-mechanical two-state model and a cooperative statistical-mechanical multistate model. The empirical model partially corrects for the deficits of the standard model. However, only the two statistical-mechanical models are thermodynamically consistent. The two-state models yield good fits for the enthalpy, entropy and free energy of unfolding of small proteins. The cooperative statistical-mechanical multistate model yields perfect fits, even for the unfolding of large proteins such as antibodies.
Topics: Protein Denaturation; Thermodynamics; Protein Unfolding; Entropy; Proteins; Calorimetry, Differential Scanning; Protein Folding
PubMed: 36982534
DOI: 10.3390/ijms24065457 -
Food Chemistry Mar 2022Physical barriers hinder about 20-25% of the protein from being extracted from whole meal. Heat-induced denaturation and aggregation of protein in quinoa seeds and in...
Physical barriers hinder about 20-25% of the protein from being extracted from whole meal. Heat-induced denaturation and aggregation of protein in quinoa seeds and in whole meal was investigated. Maximally 37% of the protein in seeds covalently aggregate when boiling for 15 min. Although embryonic cell walls surrounding protein bodies remain intact during boiling of seeds, protein aggregation is not hindered. 11S Globulin monomers first dissociate into their acidic and basic subunits which further assemble into large (> 500 kDa) mainly disulfide-linked aggregates. 2S Albumins are not involved in covalent aggregation but partially leach during seed boiling. The presence of disrupted food matrix constituents in whole meal delays denaturation and causes less aggregation of protein in whole meal than in seeds. Globulins still dissociate into their subunits but less and mainly small covalent aggregates (ca. 100-500 kDa) are formed. These novel insights allow developing new quinoa-based food products.
Topics: Chenopodium quinoa; Globulins; Hot Temperature; Protein Aggregates; Protein Denaturation; Seeds
PubMed: 34655824
DOI: 10.1016/j.foodchem.2021.131330 -
Biotechnology Progress Nov 2022To clarify the relationship between irreversible inactivation and intracellular protein denaturation of Saccharomyces pastorianus by low-pressure carbon dioxide...
To clarify the relationship between irreversible inactivation and intracellular protein denaturation of Saccharomyces pastorianus by low-pressure carbon dioxide microbubbles (CO MB) treatment, a storage test of S. pastorianus cells treated with CO MB was performed, and the effect on the intracellular protein was investigated. In the storage test, the S. pastorianus population, which decreased below the detection limit by CO MB treatment at a temperature of 45 and 50°C (MB45 and MB50), and thermal treatment at a temperature of 80°C (T80), remained undetectable during storage for 3 weeks at 25°C. However, 4.1 and 1.3-logs of the S. pastorianus populations, which survived after CO MB treatment at temperatures of 35 and 40°C (MB35 and MB40), increased gradually during storage for 3 weeks at 25°C. Insolubilization of intracellular proteins in S. pastorianus increased with increasing the temperature of CO MB treatment. Activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) identified as one of the insolubilized proteins increased at MB35 and MB40 than non-treatment but disappeared at MB45 and MB50, and T80. Therefore, it was revealed that S. pastorianus cells inactivated below the detection level by CO MB treatment did not regrow and that the denaturation of intracellular proteins of S. pastorianus was caused by CO MB and thermal treatments. Furthermore, it was suggested that denaturation of intracellular vital enzymes was an important factor for achieving irreversible inactivation of S. pastorianus by CO MB and thermal treatments.
Topics: Carbon Dioxide; Microbubbles; Protein Denaturation; Saccharomyces
PubMed: 35815350
DOI: 10.1002/btpr.3287 -
Biochemistry Feb 2022In the past, many intensive attempts failed to capture or underestimated the copopulated intermediate conformers from the protein folding/unfolding reaction. We report a...
In the past, many intensive attempts failed to capture or underestimated the copopulated intermediate conformers from the protein folding/unfolding reaction. We report a promising approach to kinetically trap, resolve, and quantify protein conformers that evolve during unfolding in solution. We conducted acid-induced unfolding of three model proteins (cytochrome , myoglobin, and lysozyme), and the corresponding reaction aliquots upon decreasing the pH were electrosprayed for high field asymmetric waveform ion mobility spectrometry (FAIMS) measurements. The copopulated conformers were resolved, visualized, and quantified by a two-dimensional mapping of the FAIMS output. Contrary to expectations, all the above proteins appeared metamorphic (multiple-folded conformations) at the physiological pH, and cytochrome exhibited an unusual "conformational shuttling" before forming the molten globule state. Thus, in contrast to many previous studies, a wide variety of thermodynamically stable intermediate conformers, including compact, molten globule, and partially unfolded forms, was trapped from solution, probing the unfolding mechanism in detail.
Topics: Cytochromes c; Hydrogen-Ion Concentration; Ion Mobility Spectrometry; Kinetics; Muramidase; Myoglobin; Protein Conformation; Protein Denaturation; Protein Folding; Thermodynamics
PubMed: 35085435
DOI: 10.1021/acs.biochem.1c00743 -
Biochimica Et Biophysica Acta. General... Feb 2020In mammalian cells, nearly one-third of proteins are inserted into the endoplasmic reticulum (ER), where they undergo oxidative folding and chaperoning assisted by... (Review)
Review
In mammalian cells, nearly one-third of proteins are inserted into the endoplasmic reticulum (ER), where they undergo oxidative folding and chaperoning assisted by approximately 20 members of the protein disulfide isomerase family (PDIs). PDIs consist of multiple thioredoxin-like domains and recognize a wide variety of proteins via highly conserved interdomain flexibility. Although PDIs have been studied intensely for almost 50 years, exactly how they maintain protein homeostasis in the ER remains unknown, and is important not only for fundamental biological understanding but also for protein misfolding- and aggregation-related pathophysiology. Herein, we review recent advances in structural biology and biophysical approaches that explore the underlying mechanism by which PDIs fulfil their distinct functions to promote productive protein folding and scavenge misfolded proteins in the ER, the primary factory for efficient production of the secretome.
Topics: Animals; Disulfides; Endoplasmic Reticulum; Humans; Membrane Glycoproteins; Mice; Mutation; Neurodegenerative Diseases; Oxidation-Reduction; Oxidative Stress; Peptides; Protein Denaturation; Protein Disulfide-Isomerases; Protein Domains; Protein Folding; Rats
PubMed: 30986509
DOI: 10.1016/j.bbagen.2019.04.003 -
Analytica Chimica Acta Feb 2023Unclear issues in protein studies include but not limited to the stability and denaturation mechanism in the presence of denaturants. Herein, we report a dynamic...
Unclear issues in protein studies include but not limited to the stability and denaturation mechanism in the presence of denaturants. Herein, we report a dynamic monitoring approach based on nanopore single-molecule biosensor, which can detect the protein's folding and unfolding transitions by recording a nanopore ionic current. When gradually increasing the concentration of denaturant guanidine hydrochloride (GdmCl), sensitive responses were observed with lysozyme unfolding. The emergence of the featured biphasic-pulse demonstrated the existence of a stable intermediate. It was the first time to experimentally confirm the dynamic equilibrium between the intermediate and the native states at single molecule level, therefore consolidating the standpoint of lysozyme denaturation process following the three-state model. Additionally, we got more insights into the conformation about the intermediate as globular-like structure, larger gyration radius, and enhanced positive charge density. We considered that the manner of denaturant toward lysozyme adopts the "direct" model based on stronger electrostatic and van der Waals forces. Nanopore biosensor exhibited excellent sensitivity with a low detection concentration of 280 pM and reproducibility in analysing the folding intermediate of lysozyme.
Topics: Protein Folding; Protein Denaturation; Muramidase; Nanopores; Reproducibility of Results; Guanidine; Kinetics; Thermodynamics; Protein Conformation
PubMed: 36697181
DOI: 10.1016/j.aca.2023.340830 -
Scientific Reports Nov 2023Thermal shift assay (TSA) with altered temperature has been the most widely used method for monitoring protein stability for drug research. However, there is a pressing...
Thermal shift assay (TSA) with altered temperature has been the most widely used method for monitoring protein stability for drug research. However, there is a pressing need for isothermal techniques as alternatives. This urgent demand arises from the limitations of TSA, which can sometimes provide misleading ranking of protein stability and fail to accurately reflect protein stability under physiological conditions. Although differential scanning fluorimetry has significantly improved throughput in comparison to differential scanning calorimetry and differential static light scattering throughput, all these methods exhibit moderate sensitivity. In contrast, current isothermal chemical denaturation (ICD) techniques may not offer the same throughput capabilities as TSA, but it provides more precise information about protein stability and interactions. Unfortunately, ICD also suffers from limited sensitivity, typically in micromolar range. We have developed a novel method to overcome these challenges, namely throughput and sensitivity. The novel Förster Resonance Energy Transfer (FRET)-Probe as an external probe is highly applicable to isothermal protein stability monitoring but also to conventional TSA. We have investigated ICD for multiple proteins with focus on KRAS with covalent inhibitors and three chemical denaturants performed at nanomolar protein concentration. Data showed corresponding inhibitor-induced stabilization of KRAS to those reported by nucleotide exchange assay.
Topics: Proto-Oncogene Proteins p21(ras); Protein Stability; Fluorometry; Calorimetry, Differential Scanning; Proteins; Protein Denaturation
PubMed: 37973851
DOI: 10.1038/s41598-023-46720-w -
FEMS Yeast Research Nov 2022This year marks the 200th anniversary of the birth of Dr Louis Pasteur (1822-1895), who revealed that alcoholic fermentation is performed by yeast cells. Subsequently,... (Review)
Review
This year marks the 200th anniversary of the birth of Dr Louis Pasteur (1822-1895), who revealed that alcoholic fermentation is performed by yeast cells. Subsequently, details of the mechanisms of alcoholic fermentation and glycolysis in yeast cells have been elucidated. However, the mechanisms underlying the high tolerance and adaptability of yeast cells to ethanol are not yet fully understood. This review presents the response and adaptability of yeast cells to ethanol-induced protein denaturation. Herein, we describe the adverse effects of severe ethanol stress on intracellular proteins and the responses of yeast cells. Furthermore, recent findings on the acquired resistance of wine yeast cells to severe ethanol stress that causes protein denaturation are discussed, not only under laboratory conditions, but also during the fermentation process at 15°C to mimic the vinification process of white wine.
Topics: Saccharomyces cerevisiae; Wine; Ethanol; Protein Denaturation; Fermentation
PubMed: 36385376
DOI: 10.1093/femsyr/foac059 -
Analytical Biochemistry Sep 2020The stability of a protein is a fundamental property that determines under which conditions, the protein is functional. Equilibrium unfolding with denaturants requires...
The stability of a protein is a fundamental property that determines under which conditions, the protein is functional. Equilibrium unfolding with denaturants requires preparation of several samples and only provides the free energy of folding when performed at a single temperature. The typical sample requirement is around 0.5-1 mg of protein. If the stability of many proteins or protein variants needs to be determined, substantial protein production may be needed. Here we have determined the stability of acyl-coenzyme A binding protein at pH 5.3 and chymotrypsin inhibitor 2 at pH 3 and pH 6.25 by combined temperature and denaturant unfolding. We used a setup where tryptophan fluorescence is measured in quartz capillaries where only 10 μl is needed. Temperature unfolding of a series of 15 samples at increasing denaturant concentrations provided accurate and precise thermodynamic parameters. We find that the number of samples may be further reduced and less than 10 μg of protein in total are needed for reliable stability measurements. For assessment of stability of protein purified in small scale e.g. in micro plate format, our method will be highly applicable. The routine for fitting the experimental data is made available as a python notebook.
Topics: Carrier Proteins; Guanidine; Kinetics; Peptides; Plant Proteins; Protein Conformation; Protein Denaturation; Protein Stability; Thermodynamics; Urea
PubMed: 32738214
DOI: 10.1016/j.ab.2020.113863