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Protein Engineering, Design & Selection... Feb 2021We review the background, theory and general equations for the analysis of equilibrium protein unfolding experiments, focusing on denaturant and heat-induced unfolding.... (Review)
Review
We review the background, theory and general equations for the analysis of equilibrium protein unfolding experiments, focusing on denaturant and heat-induced unfolding. The primary focus is on the thermodynamics of reversible folding/unfolding transitions and the experimental methods that are available for extracting thermodynamic parameters. We highlight the importance of modelling both how the folding equilibrium depends on a perturbing variable such as temperature or denaturant concentration, and the importance of modelling the baselines in the experimental observables.
Topics: Kinetics; Protein Denaturation; Protein Folding; Protein Stability; Temperature; Thermodynamics
PubMed: 33724431
DOI: 10.1093/protein/gzab002 -
Advances in Colloid and Interface... Oct 2022Although the anionic surfactant sodium dodecyl sulfate, SDS, has been used for more than half a century as a versatile and efficient protein denaturant for protein... (Review)
Review
Although the anionic surfactant sodium dodecyl sulfate, SDS, has been used for more than half a century as a versatile and efficient protein denaturant for protein separation and size estimation, there is still controversy about its mode of interaction with proteins. The term "rod-like" structures for the complexes that form between SDS and protein, originally introduced by Tanford, is not sufficiently descriptive and does not distinguish between the two current vying models, namely protein-decorated micelles a.k.a. the core-shell model (in which denatured protein covers the surface of micelles) versus beads-on-a-string model (where unfolded proteins are surrounded by surfactant micelles). Thanks to a combination of structural, kinetic and computational work particularly within the last 5-10 years, it is now possible to rule decisively in favor of the core-shell model. This is supported unambiguously by a combination of calorimetric and small-angle X-ray scattering (SAXS) techniques and confirmed by increasingly sophisticated molecular dynamics simulations. Depending on the SDS:protein ratio and the protein molecular mass, the formed structures can range from multiple partly unfolded protein molecules surrounding a single shared micelle to a single polypeptide chain decorating multiple micelles. We also have much new insight into how this species forms. It is preceded by the binding of small numbers of SDS molecules which subsequently grow by accretion. Time-resolved SAXS analysis reveals an asymmetric attack by SDS micelles followed by distribution of the increasingly unfolded protein around the micelle. The compactness of the protein chain continues to evolve at higher SDS concentrations according to single-molecule studies, though the protein remains completely denatured on the tertiary structural level. SDS denaturation can be reversed by addition of nonionic surfactants that absorb SDS forming mixed micelles, leaving the protein free to refold. Refolding can occur in parallel tracks if only a fraction of the protein is initially stripped of SDS. SDS unfolding is nearly always reversible unless carried out at low pH, where charge neutralization can lead to superclusters of protein-surfactant complexes. With the general mechanism of SDS denaturation now firmly established, it largely remains to explore how other ionic surfactants (including biosurfactants) may diverge from this path.
Topics: Micelles; Proteins; Scattering, Small Angle; Sodium Dodecyl Sulfate; Surface-Active Agents; X-Ray Diffraction
PubMed: 36027673
DOI: 10.1016/j.cis.2022.102754 -
Frontiers in Molecular Biosciences 2022Human health depends on the correct folding of proteins, for misfolding and aggregation lead to diseases. An unfolded (denatured) protein can refold to its original...
Human health depends on the correct folding of proteins, for misfolding and aggregation lead to diseases. An unfolded (denatured) protein can refold to its original folded state. How does this occur is known as the protein folding problem. One of several related questions to this problem is that how much more stable is the folded state than the unfolded state. There are several measures of protein stability. In this article, protein stability is given a thermodynamic definition and is measured by Gibbs free energy change ( ) associated with the equilibrium, native (N) conformation ↔ denatured (D) conformation under the physiological condition usually taken as dilute buffer (or water) at 25 °C. We show that this thermodynamic quantity ( ), where subscript D represents transition between N and D states, and superscript 0 (zero) represents the fact that the transition occurs in the absence of denaturant, can be neither measured nor predicted under physiological conditions. However, can be measured in the presence of strong chemical denaturants such as guanidinium chloride and urea which are shown to destroy all noncovalent interactions responsible for maintaining the folded structure. A problem with this measurement is that the estimate of comes from the analysis of the plot of denaturant concentration, which requires a long extrapolation of values of , and all the three methods of extrapolation give three different values of for a protein. Thus, our confidence in the authentic value of is eroded. Another problem with this measurement of is that it is done on the pure protein sample in dilute buffer which is a very large extrapolation of the conditions, for the crowding effect on protein stability is ignored.
PubMed: 35992266
DOI: 10.3389/fmolb.2022.880358 -
Journal of the Royal Society, Interface Oct 2018Although it is now relatively well understood how sequence defines and impacts global protein stability in specific structural contexts, the question of how sequence... (Review)
Review
Although it is now relatively well understood how sequence defines and impacts global protein stability in specific structural contexts, the question of how sequence modulates the configurational landscape of proteins remains to be defined. Protein configurational equilibria are generally characterized by using various chemical denaturants or by changing temperature or pH. Another thermodynamic parameter which is less often used in such studies is high hydrostatic pressure. This review discusses the basis for pressure effects on protein structure and stability, and describes how the unique mechanisms of pressure-induced unfolding can provide unique insights into protein conformational landscapes.
Topics: Animals; Pressure; Protein Denaturation; Protein Stability; Proteins; Thermodynamics
PubMed: 30282759
DOI: 10.1098/rsif.2018.0244 -
Yi Chuan = Hereditas Mar 2018Co-amplification at lower denaturation temperature-polymerase chain reaction (COLD-PCR) is a novel form of PCR that selectively denatures and amplifies low-abundance... (Review)
Review
Co-amplification at lower denaturation temperature-polymerase chain reaction (COLD-PCR) is a novel form of PCR that selectively denatures and amplifies low-abundance mutations from mixtures of wild-type and mutation-containing sequences, enriching the mutation 10 to 100 folds. Due to the slightly altered melting temperature (Tm) of the double-stranded DNA and the formation of the mutation/wild-type heteroduplex DNA, COLD-PCR methods are sensitive, specific, accurate, cost-effective and easy to maneuver, and can enrich mutations of any type and at any position, even unknown mutations within amplicons. COLD-PCR and its improved methods are now applied in cancer, microorganisms, prenatal screening, animals and plants. They are extremely useful for early diagnosis, monitoring the prognosis of disease and the efficiency of the treatment, drug selection, prediction of prognosis, plant breeding and etc. In this review, we introduce the principles, key techniques, derived methods and applications of COLD-PCR.
Topics: Animals; DNA Mutational Analysis; Humans; Polymerase Chain Reaction; Temperature
PubMed: 29576546
DOI: 10.16288/j.yczz.17-369 -
Biomedicines Oct 2021While protein refolding has been studied for over 50 years since the pioneering work of Christian Anfinsen, there have been a limited number of studies correlating...
While protein refolding has been studied for over 50 years since the pioneering work of Christian Anfinsen, there have been a limited number of studies correlating results between chemical, thermal, and mechanical unfolding. The limited knowledge of the relationship between these processes makes it challenging to compare results between studies if different refolding methods were applied. Our current work compares the energetic barriers and folding rates derived from chemical, thermal, and mechanical experiments using an immunoglobulin-like domain from the muscle protein titin as a model system. This domain, I83, has high solubility and low stability relative to other Ig domains in titin, though its stability can be modulated by calcium. Our experiments demonstrated that the free energy of refolding was equivalent with all three techniques, but the refolding rates exhibited differences, with mechanical refolding having slightly faster rates. This suggests that results from equilibrium-based measurements can be compared directly but care should be given comparing refolding kinetics derived from refolding experiments that used different unfolding methods.
PubMed: 34680512
DOI: 10.3390/biomedicines9101395 -
Frontiers in Neuroscience 2022Amyloid formation is linked to devastating neurodegenerative diseases, motivating detailed studies of the mechanisms of amyloid formation. For Aβ, the peptide...
Amyloid formation is linked to devastating neurodegenerative diseases, motivating detailed studies of the mechanisms of amyloid formation. For Aβ, the peptide associated with Alzheimer's disease, the mechanism and rate of aggregation have been established for a range of variants and conditions and in bodily fluids. A key outstanding question is how the relative stabilities of monomers, fibrils and intermediates affect each step in the fibril formation process. By monitoring the kinetics of aggregation of Aβ42, in the presence of urea or guanidinium hydrochloride (GuHCl), we here determine the rates of the underlying microscopic steps and establish the importance of changes in relative stability induced by the presence of denaturant for each individual step. Denaturants shift the equilibrium towards the unfolded state of each species. We find that a non-ionic denaturant, urea, reduces the overall aggregation rate, and that the effect on nucleation is stronger than the effect on elongation. Urea reduces the rate of secondary nucleation by decreasing the coverage of fibril surfaces and the rate of nucleus formation. It also reduces the rate of primary nucleation, increasing its reaction order. The ionic denaturant, GuHCl, accelerates the aggregation at low denaturant concentrations and decelerates the aggregation at high denaturant concentrations. Below approximately 0.25 M GuHCl, the screening of repulsive electrostatic interactions between peptides by the charged denaturant dominates, leading to an increased aggregation rate. At higher GuHCl concentrations, the electrostatic repulsion is completely screened, and the denaturing effect dominates. The results illustrate how the differential effects of denaturants on stability of monomer, oligomer and fibril translate to differential effects on microscopic steps, with the rate of nucleation being most strongly reduced.
PubMed: 36203800
DOI: 10.3389/fnins.2022.943355 -
Biochemistry May 2019Protein unfolding thermodynamic parameters are conventionally extracted from equilibrium thermal and chemical denaturation experiments. Despite decades of work, the...
Protein unfolding thermodynamic parameters are conventionally extracted from equilibrium thermal and chemical denaturation experiments. Despite decades of work, the degree of structure and the compactness of denatured states populated in these experiments are still open questions. Here, building on previous works, we show that thermally and chemically denatured protein states are distinct from the viewpoint of far-ultraviolet circular dichroism experiments that report on the local conformational status of peptide bonds. The differences identified are independent of protein length, structural class, or experimental conditions, highlighting the presence of two sub-ensembles within the denatured states. The sub-ensembles, U and U for thermally induced and denaturant-induced unfolded states, respectively, can exclusively exchange populations as a function of temperature at high chemical denaturant concentrations. Empirical analysis suggests that chemically denatured states are ∼50% more expanded than the thermally denatured chains of the same protein. Our observations hint that the strength of protein backbone-backbone interactions and identity versus backbone-solvent/co-solvent interactions determine the conformational distributions. We discuss the implications for protein folding mechanisms, the heterogeneity in relaxation rates, and folding diffusion coefficients.
Topics: Circular Dichroism; DNA-Binding Proteins; Escherichia coli Proteins; Hot Temperature; Kinetics; Protein Conformation, alpha-Helical; Protein Denaturation; Protein Folding; Repressor Proteins; Urea
PubMed: 31083972
DOI: 10.1021/acs.biochem.9b00089 -
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