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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 -
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 -
Physical Review Letters Mar 2006DNA adsorption and naturation is modeled via two interacting flexible homopolymers coupled to a solid surface. DNA denatures if the entropy gain for unbinding the two...
DNA adsorption and naturation is modeled via two interacting flexible homopolymers coupled to a solid surface. DNA denatures if the entropy gain for unbinding the two strands overcomes the loss of binding energy. When adsorbed to a surface, the entropy gain is smaller than in the bulk, leading to a stronger binding and, upon neglecting self-avoidance, absence of a denatured phase. Now consider conditions where the binding potentials are too weak for naturation, and the surface potential too weak to adsorb single strands. In a variational approach it is shown that their combined action may lead to a naturated adsorbed phase. Conditions for the absence of naturation and adsorption are derived too. The phase diagram is constructed qualitatively.
Topics: DNA; Models, Chemical; Nucleic Acid Denaturation; Tissue Adhesions
PubMed: 16606322
DOI: 10.1103/PhysRevLett.96.098302 -
The Journal of Biological Chemistry Nov 1997alpha-Crystallin, the major protein in the mammalian lens, is a molecular chaperone that can bind denaturing proteins and prevent their aggregation. Like other...
alpha-Crystallin, the major protein in the mammalian lens, is a molecular chaperone that can bind denaturing proteins and prevent their aggregation. Like other structurally related small heat shock proteins, each alpha-crystallin molecule is composed of an average of 40 subunits that can undergo extensive reorganization. In this study we used fluorescence resonance energy transfer to monitor the rapid exchange of recombinant alpha-crystallin subunits. We labeled alphaA-crystallin with stilbene iodoacetamide (4-acetamido-4'-((iodoacetyl)amino)stilbene-2,2'-disulfonic acid), which serves as an energy donor and with lucifer yellow iodoacetamide, which serves as an energy acceptor. Upon mixing the two populations of labeled alphaA-crystallin, we observed a reversible, time-dependent decrease in stilbene iodoacetamide emission intensity and a concomitant increase in lucifer yellow iodoacetamide fluorescence. This result is indicative of an exchange reaction that brings the fluorescent alphaA-crystallin subunits close to each other. We further showed that the exchange reaction is strongly dependent on temperature, with a rate constant of 0.075 min-1 at 37 degrees C and an activation energy of 60 kcal/mol. The subunit exchange is independent of pH and calcium concentration but decreases at low and high ionic strength, suggesting the involvement of both ionic and hydrophobic interactions. It is also markedly reduced by the binding of large denatured proteins. The degree of inhibition is directly proportional to the molecular mass and the amount of bound polypeptide, suggesting an interaction of several alphaA-crystallin subunits with multiple binding sites of the denaturing protein. Our findings reveal a dynamic organization of alphaA-crystallin subunits, which may be a key factor in preventing protein aggregation during denaturation.
Topics: Animals; Calcium; Crystallins; Hydrogen-Ion Concentration; Osmolar Concentration; Protein Conformation; Protein Denaturation; Rats; Recombinant Proteins; Sodium; Spectrometry, Fluorescence
PubMed: 9368012
DOI: 10.1074/jbc.272.47.29511 -
The Journal of Physical Chemistry... May 2019Denaturants such as the guanidinium cation unfold proteins at molar concentrations, which interferes with ultraviolet- and infrared-based spectroscopy measurements....
Denaturants such as the guanidinium cation unfold proteins at molar concentrations, which interferes with ultraviolet- and infrared-based spectroscopy measurements. Dodine denatures some proteins cooperatively at a thousand-fold lower concentration, allowing for spectroscopy measurements. Nonetheless, dodine's microscopic mechanism of interaction with proteins is not understood. We probe the effect of dodine on α-helices and tertiary structure by investigating the stability of the small helical protein B. Experiments show that dodine promotes formation of helical structure (a kosmotropic effect), while inducing the loss of tertiary structure (a chaotropic effect). Although dodine destabilizes native protein structure, it does not lower the thermal denaturation midpoint temperature of protein B. All-atom simulations reveal the cause for both observations: The denaturant action of dodine's guanidyl headgroup is counteracted by its aliphatic tail, which stabilizes amphipathic helices and associates with an expanded protein core. The Janus-like behavior of headgroup and tail make dodine a simultaneous stabilizer-destabilizer or "kosmo-chaotrope".
Topics: Guanidines; Hydrogen Bonding; Protein Conformation; Protein Denaturation; Receptors, Fc; Spectrophotometry, Infrared; Spectrophotometry, Ultraviolet
PubMed: 31026167
DOI: 10.1021/acs.jpclett.9b00379 -
BBA Advances 2022C-reactive protein (CRP) is commonly measured as an inflammatory marker in patient studies for coronary heart disease, autoimmune disease and recent acute infections....
C-reactive protein (CRP) is commonly measured as an inflammatory marker in patient studies for coronary heart disease, autoimmune disease and recent acute infections. Due to a correlation of CRP to a vast number of disease states, CRP is a well-studied protein in medical literature with over 16000 references in PubMed [1]. However, the biochemical and structural variations of CRP are not well understood in regards to their binding of complement immune response proteins. Conformations of CRP are thought to affect disease states differently, with a modified form showing neoepitopes and activating the complement immune response through C1q binding. In this work, we compare the unfolding of CRP using chemical denaturants and identify which states of CRP bind a downstream complement immune response binding partner (C1q). We used guanidine HCl (GndHCl), urea/EDTA, and 0.01% SDS with heat to perturb the pentameric state. All treatments give rise to a monomeric state in non-denaturing polyacrylamide gel electrophoresis experiments, but only treatment with certain concentrations of denaturant or dilute SDS with heat maintains CRP function with a key downstream binding partner, C1q, as measured by enzyme-linked immunosorbent assays. The results suggest that the final form of modified CRP and its ability to mimic biological binding is dependent on the preparation method.
PubMed: 37082597
DOI: 10.1016/j.bbadva.2022.100058 -
Journal of Biomolecular NMR Jan 2021Advanced NMR methods combined with biophysical techniques have recently provided unprecedented insight into structure and dynamics of molecular chaperones and their...
Advanced NMR methods combined with biophysical techniques have recently provided unprecedented insight into structure and dynamics of molecular chaperones and their interaction with client proteins. These studies showed that several molecular chaperones are able to dissolve aggregation-prone polypeptides in aqueous solution. Furthermore, chaperone-bound clients often feature fluid-like backbone dynamics and chaperones have a denaturing effect on clients. Interestingly, these effects that chaperones have on client proteins resemble the effects of known chaotropic substances. Following this analogy, chaotropicity could be a fruitful concept to describe, quantify and rationalize molecular chaperone function. In addition, the observations raise the possibility that at least some molecular chaperones might share functional similarities with chaotropes. We discuss these concepts and outline future research in this direction.
Topics: Humans; Models, Molecular; Molecular Chaperones; Nuclear Magnetic Resonance, Biomolecular; Protein Binding; Protein Conformation; Protein Denaturation; Protein Folding; Protein Unfolding; Proteins; Solubility; Structure-Activity Relationship
PubMed: 33136251
DOI: 10.1007/s10858-020-00353-7 -
International Journal of Molecular... Aug 2022Guanidinium (Gdm) undergoes interactions with both hydrophilic and hydrophobic groups and, thus, is a highly potent denaturant of biomolecular structure. However, our...
Guanidinium (Gdm) undergoes interactions with both hydrophilic and hydrophobic groups and, thus, is a highly potent denaturant of biomolecular structure. However, our molecular understanding of the interaction of Gdm with proteins and DNA is still rather limited. Here, we investigated the denaturation of DNA origami nanostructures by three Gdm salts, i.e., guanidinium chloride (GdmCl), guanidinium sulfate (GdmSO), and guanidinium thiocyanate (GdmSCN), at different temperatures and in dependence of incubation time. Using DNA origami nanostructures as sensors that translate small molecular transitions into nanostructural changes, the denaturing effects of the Gdm salts were directly visualized by atomic force microscopy. GdmSCN was the most potent DNA denaturant, which caused complete DNA origami denaturation at 50 °C already at a concentration of 2 M. Under such harsh conditions, denaturation occurred within the first 15 min of Gdm exposure, whereas much slower kinetics were observed for the more weakly denaturing salt GdmSO at 25 °C. Lastly, we observed a novel non-monotonous temperature dependence of DNA origami denaturation in GdmSO with the fraction of intact nanostructures having an intermediate minimum at about 40 °C. Our results, thus, provide further insights into the highly complex Gdm-DNA interaction and underscore the importance of the counteranion species.
Topics: DNA; Guanidine; Guanidines; Protein Denaturation; Salts; Sulfates; Thiocyanates
PubMed: 35955680
DOI: 10.3390/ijms23158547 -
Materials (Basel, Switzerland) Dec 2015Hemoglobin is a promising drug carrier but lacks extensive investigation. The chemical conjugation of hemoglobin and drugs is costly and complex, so we have developed...
Hemoglobin is a promising drug carrier but lacks extensive investigation. The chemical conjugation of hemoglobin and drugs is costly and complex, so we have developed curcumin-loaded hemoglobin nanoparticles (CCM-Hb-NPs) via self-assembly for the first time. Using the acid-denaturing method, we avoid introducing denaturants and organic solvents. The nanoparticles are stable with uniform size. We have conducted a series of experiments to examine the interaction of hemoglobin and CCM, including hydrophobic characterization, SDS-PAGE. These experiments substantiate that this self-assembly process is mainly driven by hydrophobic forces. Our nanoparticles achieve much higher cell uptake efficiency and cytotoxicity than free CCM solution . The uptake inhibition experiments also demonstrate that our nanoparticles were incorporated via the classic clathrin-mediated endocytosis pathway. These results indicate that hemoglobin nanoparticles formed by self-assembly are a promising drug delivery system for cancer therapy.
PubMed: 28793739
DOI: 10.3390/ma8125486 -
Biophysical Journal Mar 2016Denaturant-induced unfolding of helical membrane proteins provides insights into their mechanism of folding and domain organization, which take place in the chemically...
Denaturant-induced unfolding of helical membrane proteins provides insights into their mechanism of folding and domain organization, which take place in the chemically heterogeneous, anisotropic environment of a lipid membrane. Rhomboid proteases are intramembrane proteases that play key roles in various diseases. Crystal structures have revealed a compact helical bundle with a buried active site, which requires conformational changes for the cleavage of transmembrane substrates. A dimeric form of the rhomboid protease has been shown to be important for activity. In this study, we examine the mechanism of refolding for two distinct rhomboids to gain insight into their secondary structure-activity relationships. Although helicity is largely abolished in the unfolded states of both proteins, unfolding is completely reversible for HiGlpG but only partially reversible for PsAarA. Refolding of both proteins results in reassociation of the dimer, with a 90% regain of catalytic activity for HiGlpG but only a 70% regain for PsAarA. For both proteins, a broad, gradual transition from the native, folded state to the denatured, partly unfolded state was revealed with the aid of circular dichroism spectroscopy as a function of denaturant concentration, thus arguing against a classical two-state model as found for many globular soluble proteins. Thermal denaturation has irreversible destabilizing effects on both proteins, yet reveals important functional details regarding substrate accessibility to the buried active site. This concerted biophysical and functional analysis demonstrates that HiGlpG, with a simple six-transmembrane-segment organization, is more robust than PsAarA, which has seven predicted transmembrane segments, thus rendering HiGlpG amenable to in vitro studies of membrane-protein folding.
Topics: Bacterial Proteins; Chromatography, Gel; Circular Dichroism; Endopeptidases; Haemophilus influenzae; Kinetics; Membrane Proteins; Mutant Proteins; Protein Denaturation; Protein Folding; Protein Multimerization; Protein Refolding; Protein Structure, Secondary; Providencia; Temperature; Time Factors
PubMed: 27028647
DOI: 10.1016/j.bpj.2016.01.032