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Protein Expression and Purification Jul 2005Arginine is a useful solvent additive for many applications, including refolding and solubilization of proteins from insoluble pellets, and suppression of protein... (Review)
Review
Arginine is a useful solvent additive for many applications, including refolding and solubilization of proteins from insoluble pellets, and suppression of protein aggregation and non-specific adsorption during formulation and purification. However, there is a concern that arginine may be a protein-denaturant, which may limit the expansion of its applications. Such concern arises from the facts that arginine decreases melting temperature and perturbs the spectroscopic properties of certain proteins and contains a guanidinium group, which is a critical chemical structure for denaturing activity of guanidine hydrochloride. Here, we show that although arginine does lower the melting temperatures of certain proteins, the extent is insufficient to cause denaturation of proteins at or below room temperature. The proteins described here show enzymatic activity and folded structure in the presence of arginine, although the local structure around aromatic amino acids is perturbed by arginine. Arginine differs from guandinine hydrochloride in the mode of interactions with proteins, which may be a primary reason why arginine is not a protein-denaturant.
Topics: Amino Acids, Aromatic; Arginine; Guanidine; Protein Denaturation; Protein Structure, Tertiary; Surface Properties; Tryptophan; Tyrosine
PubMed: 15893471
DOI: 10.1016/j.pep.2005.03.028 -
European Biophysics Journal : EBJ Jan 2018Protein thermodynamic stability is intricately linked to cellular function, and altered stability can lead to dysfunction and disease. The linear extrapolation model...
Protein thermodynamic stability is intricately linked to cellular function, and altered stability can lead to dysfunction and disease. The linear extrapolation model (LEM) is commonly used to obtain protein unfolding free energies ([Formula: see text]) by extrapolation of solvent denaturation data to zero denaturant concentration. However, for some proteins, different denaturants result in non-coincident LEM-derived [Formula: see text] values, raising questions about the inherent assumption that the obtained [Formula: see text] values are intrinsic to the protein. Here, we used single-molecule FRET measurements to better understand such discrepancies by directly probing changes in the dimensions of the protein G B1 domain (GB1), a well-studied protein folding model, upon urea and guanidine hydrochloride denaturation. A comparison of the results for the two denaturants suggests denaturant-specific structural energetics in the GB1 denatured ensemble, revealing a role of the denatured state in the variable thermodynamic behavior of proteins.
Topics: Bacterial Proteins; Fluorescence Resonance Energy Transfer; Guanidine; Protein Denaturation; Protein Domains; Thermodynamics; Urea
PubMed: 29080139
DOI: 10.1007/s00249-017-1260-4 -
International Journal of Biological... Mar 2015Glossoscolex paulistus extracellular hemoglobin (HbGp) stability has been monitored in the presence of denaturant agents. 8-Anilino-1-naphtalene-sulfonic acid (ANS) was...
Glossoscolex paulistus extracellular hemoglobin (HbGp) stability has been monitored in the presence of denaturant agents. 8-Anilino-1-naphtalene-sulfonic acid (ANS) was used, and spectroscopic and hydrodynamic studies were developed. Dodecyltrimethylammonium bromide (DTAB) induces an increase in ANS fluorescence emission intensity, with maximum emission wavelength blue-shifted from 517 to 493 nm. Two transitions are noticed, at 2.50 and 9.50 mmol/L of DTAB, assigned to ANS interaction with pre-micellar aggregates and micelles, respectively. In oxy-HbGp, ANS binds to protein sites less exposed to solvent, as compared to DTAB micelles. In DTAB-HbGp-ANS ternary system, at pH 7.0, protein aggregation, oligomeric dissociation and unfolding were observed, while, at pH 5.0, aggregation is absent. DTAB induced unfolding process displays two transitions, one due to oligomeric dissociation and the second one, probably, to the denaturation of dissociated subunits. Moreover, guanidine hydrochloride and urea concentrations above 1.5 and 4.0 mol/L, respectively, induce the full HbGp denaturation, with reduction of ANS-bound oxy-HbGp hydrophobic patches, as noticed by fluorescence quenching up to 1.0 and 5.0 mol/L of denaturants. Our results show clearly the differences in probe sensitivity to the surfactant, in the presence and absence of protein, and new insights into the denaturant effects on HbGp unfolding.
Topics: Anilino Naphthalenesulfonates; Animals; Hemoglobins; Hydrodynamics; Hydrogen-Ion Concentration; Oligochaeta; Oxygen; Protein Denaturation; Protein Multimerization; Protein Stability
PubMed: 25546245
DOI: 10.1016/j.ijbiomac.2014.12.035 -
Journal of the American Chemical Society Sep 2016Chemical denaturants are the most commonly used agents for unfolding proteins and are thought to act by better solvating the unfolded state. Improved solvation is...
Chemical denaturants are the most commonly used agents for unfolding proteins and are thought to act by better solvating the unfolded state. Improved solvation is expected to lead to an expansion of unfolded chains with increasing denaturant concentration, providing a sensitive probe of the denaturant action. However, experiments have so far yielded qualitatively different results concerning the effects of chemical denaturation. Studies using Förster resonance energy transfer (FRET) and other methods found an increase in radius of gyration with denaturant concentration, but most small-angle X-ray scattering (SAXS) studies found no change. This discrepancy therefore challenges our understanding of denaturation mechanism and more generally the accuracy of these experiments as applied to unfolded or disordered proteins. Here, we use all-atom molecular simulations to investigate the effect of urea and guanidinium chloride on the structure of the intrinsically disordered protein ACTR, which can be studied by experiment over a wide range of denaturant concentration. Using unbiased molecular simulations with a carefully calibrated denaturant model, we find that the protein chain indeed swells with increasing denaturant concentration. This is due to the favorable association of urea or guanidinium chloride with the backbone of all residues and with the side-chains of almost all residues, with denaturant-water transfer free energies inferred from this association in reasonable accord with experimental estimates. Interactions of the denaturants with the backbone are dominated by hydrogen bonding, while interactions with side-chains include other contributions. By computing FRET efficiencies and SAXS intensities at each denaturant concentration, we show that the simulation trajectories are in accord with both experiments on this protein, demonstrating that there is no fundamental inconsistency between the two types of experiment. Agreement with experiment also supports the picture of chemical denaturation described in our simulations, driven by weak association of denaturant with the protein. Our simulations support some assumptions needed for each experiment to accurately reflect changes in protein size, namely, that the commonly used FRET chromophores do not qualitatively alter the results and that possible effects such as preferential solvent partitioning into the interior of the chain do not interfere with the determination of radius of gyration from the SAXS experiments.
Topics: Intrinsically Disordered Proteins; Molecular Dynamics Simulation; Protein Conformation; Protein Denaturation; Urea
PubMed: 27583687
DOI: 10.1021/jacs.6b05443 -
Journal of Biomolecular Structure &... Feb 2022Osmolytes are known to stabilize proteins against denaturing conditions. Ethylene glycol (EG), however, shows a distinctive effect on α-lactalbumin (α-LA) that it...
Osmolytes are known to stabilize proteins against denaturing conditions. Ethylene glycol (EG), however, shows a distinctive effect on α-lactalbumin (α-LA) that it stabilizes the protein against cold-induced denaturation, whereas it destabilizes during heat denaturation. The replica exchange molecular dynamics (REMD) simulation of α-LA in the presence of EG shows that EG denatures the protein at higher temperatures whereas it retards the denaturation at sub-zero temperature. Representative structures of α-LA were selected from REMD trajectories at three different temperature conditions (240, 300 and 340 K) with and without EG, and classical molecular dynamics (MD) simulations were performed. The results suggest that the presence of water around α-LA is more at lower temperatures; however, water around the hydrophobic residues is reduced with the addition of EG at sub-zero temperature. The partition coefficient of EG showed that the binding of EG with hydrophobic residues was higher at lower temperatures. Preferential interaction parameters at different temperatures were calculated based on the mean distribution (Γ) and Kirkwood-Buff integral () methods. Γ shows a larger positive value at 240 K compared to higher temperatures. shows positive values at lower temperatures, whereas it becomes negative at above 280 K. These results indicate that the preferential binding of EG with α-LA is more at sub-zero temperature compared to higher temperature conditions. Thus, the study suggests that the preferential binding of EG reduces the hydrophobic hydration of α-LA at lower temperatures, and stabilizes the protein against cold denaturation. However, the preferential binding of EG at higher temperature drives the folding equilibrium towards the denatured state.Communicated by Ramaswamy H. Sarma.
Topics: Ethylene Glycol; Lactalbumin; Molecular Dynamics Simulation; Protein Denaturation; Protein Folding; Protein Stability; Temperature; Thermodynamics
PubMed: 32954952
DOI: 10.1080/07391102.2020.1819422 -
Biochemistry Nov 2009Conformational properties of the folded and unfolded ensembles of human interleukin-1 receptor antagonist (IL-1ra) are strongly denaturant-dependent as evidenced by...
Conformational properties of the folded and unfolded ensembles of human interleukin-1 receptor antagonist (IL-1ra) are strongly denaturant-dependent as evidenced by high-resolution two-dimensional nuclear magnetic resonance (NMR), limited proteolysis, and small-angle X-ray scattering (SAXS). The folded ensemble was characterized in detail in the presence of different urea concentrations by (1)H-(15)N HSQC NMR. The beta-trefoil fold characteristic of native IL-1ra was preserved until the unfolding transition region beginning at 4 M urea. At the same time, a subset of native resonances disappeared gradually starting at low denaturant concentrations, indicating noncooperative changes in the folded state. Additional evidence of structural perturbations came from the chemical shift analysis, nonuniform and bell-shaped peak intensity profiles, and limited proteolysis. In particular, the following nearby regions of the tertiary structure became progressively destabilized with increasing urea concentrations: the beta-hairpin interface of trefoils 1 and 2 and the H2a-H2 helical region. These regions underwent small-scale perturbations within the native baseline region in the absence of populated molten globule-like states. Similar regions were affected by elevated temperatures known to induce irreversible aggregation of IL-1ra. Further evidence of structural transitions invoking near-native conformations came from an optical spectroscopy analysis of its single-tryptophan variant W17A. The increase in the radius of gyration was associated with a single equilibrium unfolding transition in the case of two different denaturants, urea and guanidine hydrochloride (GuHCl). However, the compactness of urea- and GuHCl-unfolded molecules was comparable only at high denaturant concentrations and deviated under less denaturing conditions. Our results identified the role of conformational flexibility in IL-1ra aggregation and shed light on the nature of structural transitions within the folded ensembles of other beta-trefoil proteins, such as IL-1beta and hFGF-1.
Topics: Circular Dichroism; Endopeptidase K; Fluorescence Polarization; Guanidine; Humans; Interleukin 1 Receptor Antagonist Protein; Models, Molecular; Nuclear Magnetic Resonance, Biomolecular; Protein Conformation; Protein Denaturation; Recombinant Proteins; Scattering, Radiation; Spectrometry, Fluorescence; Thermodynamics; Urea; X-Rays
PubMed: 19839644
DOI: 10.1021/bi901570k -
Protein and Peptide Letters Mar 2014In this mini-review we introduce and discuss a new method, at single molecule level, to study the protein folding and protein stability, with a nanopore coupled to an... (Review)
Review
In this mini-review we introduce and discuss a new method, at single molecule level, to study the protein folding and protein stability, with a nanopore coupled to an electric detection. Proteins unfolded or partially folded passing through one channel submitted to an electric field, in the presence of salt solution, induce different detectable blockades of ionic current. Their duration depends on protein conformation. For different studies proteins through nanopores, completely unfolded proteins induce only short current blockades. Their frequency increases as the concentration of denaturing agent or temperature increases, following a sigmoidal denaturation curve. The geometry or the net charge of the nanopores does not alter the unfolding transition, sigmoidal unfolding curve and half denaturing concentration or half temperature denaturation. A destabilized protein induces a shift of the unfolding curve towards the lower values of the denaturant agent compared to the wild type protein.Partially folded proteins exhibit very long blockades in nanopores. The blockade duration decreases when the concentration of denaturing agent increases. The variation of these blockades could be associated to a possible glassy behaviour.
Topics: Animals; Biophysics; Humans; Models, Molecular; Nanopores; Protein Conformation; Protein Denaturation; Protein Stability; Protein Unfolding; Proteins
PubMed: 24370253
DOI: 10.2174/09298665113209990080 -
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 Molecular Biology Jan 2022Functional bacterial amyloids (FuBA) are intrinsically disordered proteins (IDPs) which rapidly and efficiently aggregate, forming extremely stable fibrils. The...
Functional bacterial amyloids (FuBA) are intrinsically disordered proteins (IDPs) which rapidly and efficiently aggregate, forming extremely stable fibrils. The conversion from IDP to amyloid is evolutionarily optimized and likely couples folding to association. Many FuBA contain several imperfect repeat sequences which contribute to the stability of mature FuBA fibrils. Aggregation can be considered an intermolecular extension of the process of intramolecular protein folding which has traditionally been studied using chemical denaturants. Here we employ denaturants to investigate folding steps during fibrillation of CsgA and FapC. We quantify protein compactification (i.e. the extent of burial of otherwise exposed surface area upon association of proteins) during different stages of fibrillation based on the dependence of fibrillation rate constants on the denaturant concentration (m-values) determined from fibrillation curves. For both proteins, urea mainly affects nucleation and elongation (not fragmentation), consistent with the fact that these steps involve both intra- and intermolecular association. The two steps have similar m-values, indicating that activation steps in nucleation and elongation involve the same level of folding. Surprisingly, deletion of two or three repeats from FapC leads to larger m-values (i.e. higher compactification) during the activation step of fibril growth. This observation is extended by SAXS analysis of the fibrils which indicates that weakening of the amyloidogenic core caused by repeat deletions causes a larger portion of normally unstructured regions of the protein to be included into the amyloid backbone. We conclude that the sensitivity of fibrillation to denaturants can provide useful insight into molecular mechanisms of aggregation.
Topics: Amyloid; Amyloidogenic Proteins; Bacteria; Benzothiazoles; Escherichia coli Proteins; Intrinsically Disordered Proteins; Protein Aggregates; Protein Folding
PubMed: 34748745
DOI: 10.1016/j.jmb.2021.167337 -
Scientific Reports Jan 2021Substance use disorders are a significant public health issue. Options to dispose of controlled medications are limited, increasing the risk of diversion. Providing an...
Substance use disorders are a significant public health issue. Options to dispose of controlled medications are limited, increasing the risk of diversion. Providing an alternative for disposal, a chemical denaturant, SafeMedWaste, was designed to destroy controlled substances irreversibly and safely be placed in non-hazardous landfills. Via HPLC-MS, four formulations of SafeMedWaste were tested with 34 different liquid controlled medications from DEA schedules I-V. Beta testing assessed the efficacy of SafeMedWaste in a clinical setting and on waste generated in a manufacturing setting. Furthermore, a formulation of SafeMedWaste was tested on solid controlled medications. All 34 of the liquid medications tested (e.g., amphetamine, diazepam, fentanyl, ketamine) were fully destroyed in SafeMedWaste within 2-24 h. Analysis of a beta test sample of SafeMedWaste containing fentanyl, midazolam, and morphine waste collected in a hospital showed full denaturation of these drugs in 24 h. Variants of SafeMedWaste were optimized to denature six different controlled substance waste samples from a manufacturing facility. In contrast to side-by-side studies with a charcoal disposal system using the same drugs, SafeMedWaste fully inactivated and destroyed the controlled substances in the waste streams. Another formulation of SafeMedWaste was tested on solid medications, which were fully denatured in 48-72 h. In conclusion, SafeMedWaste irreversibly denatures controlled medications that present a problem in our society.
PubMed: 33441864
DOI: 10.1038/s41598-020-80388-w