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Biochip Journal 2022Nucleic acid testing (NAT) is important for the identification and quantification of specific nucleic acid targets, both DNA and RNA, in life sciences and clinical...
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Nucleic acid testing (NAT) is important for the identification and quantification of specific nucleic acid targets, both DNA and RNA, in life sciences and clinical diagnostics. Nucleic acid amplification can be a time-consuming step in NAT using the polymerase chain reaction (PCR) assay. Therefore, this study aimed to develop a simple method to reduce the amplification time while maintaining the PCR system. The three-step process of a general qPCR was reduced to a two-step process. The annealing/extension temperatures were increased to minimize the differences between the denaturation temperature and the annealing/extension temperatures. Subsequently, the time for each of these steps was reduced and, finally, the denaturation temperature was lowered. Taq polymerase was replaced with SD polymerase because it has strand displacement activity and is efficient in amplifying partial dsDNA at lower denaturation temperatures. In the two-step qPCR of genomic DNA using SD polymerase, the final conditions included an initial denaturation at 92 °C for 2 min, and 1 s at each cycling step with a denaturation temperature of 87 °C and an annealing/extension temperature of 72 °C. Amplification of the nucleocapsid () gene of SARS-CoV-2 RNA virus was evaluated at a template concentration as low as 10 copies. This method, named SF-qPCR (strand displacement-based fast quantitative polymerase chain reaction), can stably detect less than 10 copies of DNA and RNA within 25-40 min. This new protocol allows for sensitive and rapid detection of important DNA and RNA targets in clinical diagnosis.
SUPPLEMENTARY INFORMATION
The online version contains supplementary material available at 10.1007/s13206-021-00044-x.
PubMed: 35096279
DOI: 10.1007/s13206-021-00044-x -
Nature Communications Jul 2020Synthetic polymers are thoroughly embedded in the modern society and their consumption grows annually. Efficient routes to their production and processing have never...
Synthetic polymers are thoroughly embedded in the modern society and their consumption grows annually. Efficient routes to their production and processing have never been more important. In this respect, silk protein fibrillation is superior to conventional polymer processing, not only by achieving outstanding physical properties of materials, such as high tensile strength and toughness, but also improved process energy efficiency. Natural silk solidifies in response to flow of the liquid using conformation-dependent intermolecular interactions to desolvate (denature) protein chains. This mechanism is reproduced here by an aqueous poly(ethylene oxide) (PEO) solution, which solidifies at ambient conditions when subjected to flow. The transition requires that an energy threshold is exceeded by the flow conditions, which disrupts a protective hydration shell around polymer molecules, releasing them from a metastable state into the thermodynamically favoured crystalline state. This mechanism requires vastly lower energy inputs and demonstrates an alternative route for polymer processing.
PubMed: 32632091
DOI: 10.1038/s41467-020-17167-8 -
The Journal of Physical Chemistry. B Aug 2022The intrinsically disordered DNA-binding domain of cytidine repressor (CytR-DBD) folds in the presence of target DNA and regulates the expression of multiple genes in ....
The intrinsically disordered DNA-binding domain of cytidine repressor (CytR-DBD) folds in the presence of target DNA and regulates the expression of multiple genes in . To explore the conformational rearrangements in the unbound state and the target recognition mechanisms of CytR-DBD, we carried out single-molecule Förster resonance energy transfer (smFRET) measurements. The smFRET data of CytR-DBD in the absence of DNA show one major and one minor population assignable to an expanded unfolded state and a compact folded state, respectively. The population of the folded state increases and decreases upon titration with salt and denaturant, respectively, in an apparent two-state manner. The peak FRET efficiencies of both the unfolded and folded states change continuously with denaturant concentration, demonstrating the intrinsic flexibility of the DNA-binding domain and the deviation from a strict two-state transition. Remarkably, the CytR-DBD exhibits a compact structure when bound to both the specific and nonspecific DNA; however, the peak FRET efficiencies of the two structures are slightly but consistently different. The observed conformational heterogeneity highlights the potential structural changes required for CytR to bind variably spaced operator sequences.
Topics: DNA; Escherichia coli; Escherichia coli Proteins; Fluorescence Resonance Energy Transfer; Repressor Proteins; Spectrometry, Fluorescence
PubMed: 35969476
DOI: 10.1021/acs.jpcb.2c02791 -
International Journal of Molecular... Jun 2021Proteases are a group of enzymes with a catalytic function to hydrolyze peptide bonds of proteins. Proteases regulate the activity, signaling mechanism, fate, and...
Proteases are a group of enzymes with a catalytic function to hydrolyze peptide bonds of proteins. Proteases regulate the activity, signaling mechanism, fate, and localization of many proteins, and their dysregulation is associated with various pathological conditions. Proteases have been identified as biomarkers and potential therapeutic targets for multiple diseases, such as acquired immunodeficiency syndrome, cardiovascular diseases, osteoporosis, type 2 diabetes, and cancer, where they are essential to disease progression. Thus, protease inhibitors and inhibitor-like molecules are interesting drug candidates. To study proteases and their substrates and inhibitors, simple, rapid, and sensitive protease activity assays are needed. Existing fluorescence-based assays enable protease monitoring in a high-throughput compatible microtiter plate format, but the methods often rely on either molecular labeling or synthetic protease targets that only mimic the hydrolysis site of the true target proteins. Here, we present a homogenous, label-free, and time-resolved luminescence utilizing the protein-probe method to assay proteases with native and denatured substrates at nanomolar sensitivity. The developed protein-probe method is not restricted to any single protein or protein target class, enabling digestion and substrate fragmentation studies with the natural unmodified substrate proteins. The versatility of the assay for studying protease targets was shown by monitoring the digestion of a substrate panel with different proteases. These results indicate that the protein-probe method not only monitors the protease activity and inhibition, but also studies the substrate specificity of individual proteases.
Topics: Enzyme Assays; Peptide Hydrolases; Protein Denaturation; Proteins; Substrate Specificity; Temperature
PubMed: 34198602
DOI: 10.3390/ijms22126362 -
Journal of Chemical Theory and... Jan 2022The denaturant dependence of hydrogen-deuterium exchange (HDX) is a powerful measurement to identify the breaking of individual H-bonds and map the free energy surface...
The denaturant dependence of hydrogen-deuterium exchange (HDX) is a powerful measurement to identify the breaking of individual H-bonds and map the free energy surface (FES) of a protein including the very rare states. Molecular dynamics (MD) can identify each partial unfolding event with atomic-level resolution. Hence, their combination provides a great opportunity to test the accuracy of simulations and to verify the interpretation of HDX data. For this comparison, we use , our new and extremely fast MD package that is capable of folding proteins with an accuracy comparable to that of all-atom methods. The FESs of two naturally occurring and two designed proteins are so generated and compared to our NMR/HDX data. We find that 's accuracy is considerably improved upon modifying the energy function using a new machine-learning procedure that trains for proper protein behavior including realistic denatured states in addition to stable native states. The resulting increase in cooperativity is critical for replicating the HDX data and protein stability, indicating that we have properly encoded the underlying physiochemical interactions into an MD package. We did observe some mismatch, however, underscoring the ongoing challenges faced by simulations in calculating accurate FESs. Nevertheless, our ensembles can identify the properties of the fluctuations that lead to HDX, whether they be small-, medium-, or large-scale openings, and can speak to the breadth of the native ensemble that has been a matter of debate.
Topics: Deuterium Exchange Measurement; Entropy; Hydrogen; Protein Conformation; Proteins
PubMed: 34936354
DOI: 10.1021/acs.jctc.1c00960 -
Life (Basel, Switzerland) Jul 2021The conformational stability of globular proteins is strongly influenced by the addition to water of different co-solutes. Some of the latter destabilize the native...
The conformational stability of globular proteins is strongly influenced by the addition to water of different co-solutes. Some of the latter destabilize the native state, while others stabilize it. It is emerging that stabilizing agents are able to counteract the action of destabilizing agents. We have already provided experimental evidence that this counteraction is a general phenomenon and offered a rationalization. In the present work, we show that four different sugars, namely fructose, glucose, sucrose, and trehalose, counteract the effect of urea, tetramethylurea, sodium perchlorate, guanidinium chloride, and guanidinium thiocyanate despite the chemical and structural differences of those destabilizing agents. The rationalization we provide is as follows: (a) the solvent-excluded volume effect, a purely entropic effect, stabilizes the native state, whose solvent-accessible surface area is smaller than the one of denatured conformations; (b) the magnitude of the solvent-excluded volume effect increases markedly in ternary solutions because the experimental density of such solutions is larger than that of pure water.
PubMed: 34357025
DOI: 10.3390/life11070652 -
The Journal of Physical Chemistry. B Dec 2019Thermal and chemical unfolding of lysozyme in the presence of the guanidine HCl denaturant is a model system to compare the conventional two-state model of protein...
Thermal and chemical unfolding of lysozyme in the presence of the guanidine HCl denaturant is a model system to compare the conventional two-state model of protein unfolding with the multistate Zimm-Bragg theory. The two-state model is shown to be the noncooperative limit of the Zimm-Bragg theory. In particular, the Zimm-Bragg theory provides a molecular interpretation of the empirical linear extrapolation method (LEM) of the two-state model. Differential scanning calorimetry (DSC) experiments reported in the literature are analyzed with both methods. Lysozyme unfolding is associated with a large endothermic enthalpy that decreases significantly upon addition of guanidine HCl. In contrast, the Gibbs free energy of unfolding is small, negative, and independent of the guanidine HCl concentration, contradicting, in part, the conclusions of the LEM. The unfolding enthalpy is compensated by an even larger entropy term. The multistate Zimm-Bragg theory predicts a larger conformational enthalpy and a smaller Gibbs free energy than the two-state model. The Zimm-Bragg theory provides the protein cooperativity parameter, the average length of independently folding protein domains, and the Gibbs free energy of unfolding of individual amino acid residues. Guanidine HCl binding to lysozyme is exothermic and counteracts the endothermic unfolding enthalpy. The number of bound denaturant molecules is determined from the decrease in enthalpy and is extrapolated to the guanidine HCl-to-amino acid stoichiometry at complete lysozyme unfolding. Chemical unfolding isotherms measured with circular dichroism (CD) spectroscopy are analyzed with both models. The chemical Zimm-Bragg theory is a cooperative molecular model, yielding the guanidine HCl binding constant and the protein cooperativity parameter. It allows a quantitative comparison between thermal and chemical protein unfolding. The two reactions have almost identical changes in Gibbs free energy. However, thermal unfolding is significantly more cooperative than chemical unfolding. Finally, distinct differences are observed in thermal unfolding between DSC and CD spectroscopy.
Topics: Amino Acids; Animals; Binding Sites; Chickens; Egg White; Guanidine; Models, Chemical; Muramidase; Protein Binding; Protein Folding; Protein Unfolding; Thermodynamics
PubMed: 31686511
DOI: 10.1021/acs.jpcb.9b08816 -
Proceedings of the National Academy of... Aug 2021The cosolvent effect arises from the interaction of cosolute molecules with a protein and alters the equilibrium between native and unfolded states. Denaturants shift...
The cosolvent effect arises from the interaction of cosolute molecules with a protein and alters the equilibrium between native and unfolded states. Denaturants shift the equilibrium toward the latter, while osmolytes stabilize the former. The molecular mechanism whereby cosolutes perturb protein stability is still the subject of considerable debate. Probing the molecular details of the cosolvent effect is experimentally challenging as the interactions are very weak and transient, rendering them invisible to most conventional biophysical techniques. Here, we probe cosolute-protein interactions by means of NMR solvent paramagnetic relaxation enhancement together with a formalism we recently developed to quantitatively describe, at atomic resolution, the energetics and dynamics of cosolute-protein interactions in terms of a concentration normalized equilibrium average of the interspin distance, [Formula: see text], and an effective correlation time, τ The system studied is the metastable drkN SH3 domain, which exists in dynamic equilibrium between native and unfolded states, thereby permitting us to probe the interactions of cosolutes with both states simultaneously under the same conditions. Two paramagnetic cosolute denaturants were investigated, one neutral and the other negatively charged, differing in the presence of a carboxyamide group versus a carboxylate. Our results demonstrate that attractive cosolute-protein backbone interactions occur largely in the unfolded state and some loop regions in the native state, electrostatic interactions reduce the [Formula: see text] values, and temperature predominantly impacts interactions with the unfolded state. Thus, destabilization of the native state in this instance arises predominantly as a consequence of interactions of the cosolutes with the unfolded state.
Topics: Animals; Drosophila Proteins; Drosophila melanogaster; Models, Molecular; Protein Denaturation; Protein Folding; Protein Unfolding; Solvents; Thermodynamics; src Homology Domains
PubMed: 34404723
DOI: 10.1073/pnas.2112021118 -
Applied and Environmental Microbiology Feb 2021Acute severe ethanol stress (10% [vol/vol]) damages proteins and causes the intracellular accumulation of insoluble proteins in On the other hand, a pretreatment with...
Acute severe ethanol stress (10% [vol/vol]) damages proteins and causes the intracellular accumulation of insoluble proteins in On the other hand, a pretreatment with mild stress increases tolerance to subsequent severe stress, which is called acquired stress resistance. It currently remains unclear whether the accumulation of insoluble proteins under severe ethanol stress may be mitigated by increasing protein quality control (PQC) activity in cells pretreated with mild stress. In the present study, we examined the induction of resistance to severe ethanol stress in PQC and confirmed that a pretreatment with 6% (vol/vol) ethanol or mild thermal stress at 37°C significantly reduced insoluble protein levels and the aggregation of Lsg1, which is prone to denaturation and aggregation by stress, in yeast cells under 10% (vol/vol) ethanol stress. The induction of this stress resistance required the new synthesis of proteins; the expression of proteins comprising the bichaperone system (Hsp104, Ssa3, and Fes1), Sis1, and Hsp42 was upregulated during the pretreatment and maintained under subsequent severe ethanol stress. Since the pretreated cells of deficient mutants in the bichaperone system (Δ Δ and Δ Δ Δ) failed to sufficiently reduce insoluble protein levels and Lsg1 aggregation, the enhanced activity of the bichaperone system appears to be important for the induction of adequate stress resistance. In contrast, the importance of proteasomes and aggregases (Btn2 and Hsp42) in the induction of stress resistance has not been confirmed. These results provide further insights into the PQC activity of yeast cells under severe ethanol stress, including the brewing process. Although the budding yeast , which is used in the production of alcoholic beverages and bioethanol, is highly tolerant of ethanol, high concentrations of ethanol are also stressful to the yeast and cause various adverse effects, including protein denaturation. A pretreatment with mild stress improves the ethanol tolerance of yeast cells; however, it currently remains unclear whether it increases PQC activity and reduces the levels of denatured proteins. In the present study, we found that a pretreatment with mild ethanol upregulated the expression of proteins involved in PQC and mitigated the accumulation of insoluble proteins, even under severe ethanol stress. These results provide novel insights into ethanol tolerance and the adaptive capacity of yeast. They may also contribute to research on the physiology of yeast cells during the brewing process, in which the concentration of ethanol gradually increases.
Topics: Ethanol; Hot Temperature; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Stress, Physiological
PubMed: 33361368
DOI: 10.1128/AEM.02353-20 -
International Journal of Molecular... Jun 2021Human phenylalanine hydroxylase (PAH) is a metabolic enzyme involved in the catabolism of L-Phe in liver. Loss of conformational stability and decreased enzymatic...
Human phenylalanine hydroxylase (PAH) is a metabolic enzyme involved in the catabolism of L-Phe in liver. Loss of conformational stability and decreased enzymatic activity in PAH variants result in the autosomal recessive disorder phenylketonuria (PKU), characterized by developmental and psychological problems if not treated early. One current therapeutic approach to treat PKU is based on pharmacological chaperones (PCs), small molecules that can displace the folding equilibrium of unstable PAH variants toward the native state, thereby rescuing the physiological function of the enzyme. Understanding the PAH folding equilibrium is essential to develop new PCs for different forms of the disease. We investigate here the urea and the thermal-induced denaturation of full-length PAH and of a truncated form lacking the regulatory and the tetramerization domains. For either protein construction, two distinct transitions are seen in chemical denaturation followed by fluorescence emission, indicating the accumulation of equilibrium unfolding intermediates where the catalytic domains are partly unfolded and dissociated from each other. According to analytical centrifugation, the chemical denaturation intermediates of either construction are not well-defined species but highly polydisperse ensembles of protein aggregates. On the other hand, each protein construction similarly shows two transitions in thermal denaturation measured by fluorescence or differential scanning calorimetry, also indicating the accumulation of equilibrium unfolding intermediates. The similar temperatures of mid denaturation of the two constructions, together with their apparent lack of response to protein concentration, indicate the catalytic domains are unfolded in the full-length PAH thermal intermediate, where they remain associated. That the catalytic domain unfolds in the first thermal transition is relevant for the choice of PCs identified in high throughput screening of chemical libraries using differential scanning fluorimetry.
Topics: Binding Sites; Calorimetry, Differential Scanning; Catalytic Domain; Humans; Molecular Docking Simulation; Molecular Dynamics Simulation; Phenylalanine Hydroxylase; Phenylketonurias; Protein Conformation; Protein Denaturation; Protein Folding; Protein Stability; Temperature; Thermodynamics; Urea
PubMed: 34207146
DOI: 10.3390/ijms22126539