Search Results (9)

By performing differential scanning calorimetry(DSC) measurements on RNase A, we studied the stabilization provided by the addition of potassium aspartate(KAsp) or potassium glutamate (KGlu) and found that it leads to a significant increase in the denaturation temperature of the protein. The stabilization proves to be mainly entropic in origin. A counteraction of the stabilization provided by KAsp or KGlu is obtained by adding common denaturants such as urea, guanidinium chloride, or guanidinium thiocyanate. A rationalization of the experimental data is devised on the basis of a theoretical approach developed by one of the authors. The main contribution to the conformational stability of globular proteins comes from the gain in translational entropy of water and co-solute ions and/or molecules for the decrease in solvent-excluded volume associated with polypeptide folding (i.e., there is a large decrease in solvent-accessible surface area). The magnitude of this entropic contribution increases with the number density and volume packing density of the solution. The two destabilizing contributions come from the conformational entropy of the chain, which should not depend significantly on the presence of co-solutes, and from the direct energetic interactions between co-solutes and the protein surface in both the native and denatured states. It is the magnitude of the latter that discriminates between stabilizing and destabilizing agents. Full article

Peripheral blood is the most practical tissue for human immune system gene expression profiling because it is easily accessible, whereas the site of primary infection in certain diseases may not be easily accessible. Due to the ex vivo instability of RNA transcripts, a key challenge in the gene expression analysis of blood samples is the rapid sample handling and stabilization of the mRNA by adding an RNA preservative (PAXgene^TM Blood RNA Tubes, Tempus^TM Blood RNA tubes, RNAlater Stabilization Reagent, RNAgard^® Blood Tubes). BioMole (Turin, Italy) has developed a novel blood stabilizer, called RNApro, in which RNA is stabilized during phlebotomy and sample storage. In this study, RNApro performance intended as RNA yield, integrity, and stability was evaluated. Our results show that blood samples stored at −80 °C and re-extracted after 7 years show no differences in terms of quantity, quality, and amplificability. The samples in the RNAlater stabilization solution can be stored at room temperature for up to one week or at 4 °C for up to one month. Similar results can also be observed for PAXgene tubes, Tempus tubes, and RNAgard tubes. In agreement with these data, the RNApro stabilization solution preserves the RNA from degradation for up to 1 month at 4 °C and 1 week at room temperature. RNApro can be stored indifferently at −80, −20, 4 °C, or room temperature for up to 2 months after, and then could be stored at −80 °C for up to seven years. In summary, our study is the first to analyze the performance of an RNA stabilizer called RNApro. We can conclude that several studies have shown significant differences in gene expression analysis when the sample was preserved in different RNA stabilizers. Therefore, it is desirable to standardize the method of nucleic acid conservation when comparing data from transcriptomic analyses. Full article

An exhaustive analysis of all the protein structures deposited in the Protein Data Bank, here performed, has allowed the identification of hundredths of protein-bound urea molecules and the structural characterization of such binding sites. It emerged that, even though urea molecules are largely involved in hydrogen bonds with both backbone and side chains, they are also able to make van der Waals contacts with nonpolar moieties. As similar findings have also been previously reported for guanidinium and thiocyanate, this observation suggests that promiscuity is a general property of protein denaturants. Present data provide strong support for a mechanism based on the protein-denaturant direct interactions with a denaturant binding model to equal and independent sites. In this general framework, our investigations also highlight some interesting insights into the different denaturing power of urea compared to guanidinium/thiocyanate. Full article

Isolating DNA from bacterial cells concentrated directly from water samples allows the analysis of the DNA with a range of molecular biology techniques. The aim was to develop a cost-effective method to concentrate bacterial cells directly from water for DNA extraction and PCR amplification. A modified in-house guanidinium thiocyanate DNA extraction method was compared to four commercial kits (two repeats performed in triplicate) from 10-fold serially diluted bacterial cells and used to construct standard curves using quantitative real-time PCR (q-PCR). The in-house DNA extraction method-constructed qPCR standard curves showed similar results with determination coefficient (R²) of 0.99 and 0.99 and of slopes −3.48 and −3.65). The R² and slope for Water Master^TM DNA purification kit (R² 0.34, 0.73; slope −5.73, −4.45); Ultra Clean^TM Water DNA isolation kit (R² 0.97, 0.28; slope −3.89, −8.84); Aquadien^TM kit (R² 0.98, 0.77; slope −3.59, −5.94) and Metagenomic DNA isolation kit (R² 0.65, 0.77; slope −3.83, −4.89) showed higher variability than the in-house DNA extraction method. The results showed that the in-house DNA extraction method is a viable cost-effective alternative with good DNA recovery and repeatable and reproducible results. A limitation of the study is the limited number of repeats, due to cost implication of the commercial kits. Full article

Red fluorescent proteins with a large Stokes’ shift (LSSRFPs) are genetically encoded and efficiently excited by 488 nm light, allowing simultaneous dual-color one- and two-photon fluorescence imaging and fluorescence correlation spectroscopy in combination with green fluorescent proteins FPs. Recently, based on the conventional bright mScarlet RFP, we developed the LSSRFP LSSmScarlet. LSSmScarlet is characterized by two pKa values at pH values of 1.9 and 5.8. In this study, we developed improved versions of LSSmScarlet, named LSSmScarlet2 and LSSmScarlet3, which are characterized by a Stokes’ shift of 128 nm and extreme pH stability with a single pKa value of 2.2. LSSmScarlet2 and LSSmScarlet3 had 1.8-fold faster and 3-fold slower maturation than LSSmScarlet, respectively. In addition, both LSSRFPs were 1.5- to 1.6-fold more photostable and more chemically resistant to denaturation by guanidinium chloride and guanidinium thiocyanate. We also compared the susceptibility of the LSSmScarlet2, LSSmScarlet3, and other LSSRFPs to the reagents used for whole-mount imaging, expansion microscopy, and immunostaining techniques. Due to higher pH stability and faster maturation, the LSSmScarlet3-LAMP3 fusion was 2.2-fold brighter than LSSmScarlet-LAMP3 in lysosomes of mammalian cells. The LSSmScarlet3-hLAMP2A fusion was similar in brightness to LSSmScarlet-hLAMP2A in lysosomes. We successfully applied the monomeric LSSmScarlet2 and LSSmScarlet3 proteins for confocal imaging of structural proteins in live mammalian cells. We also solved the X-ray structure of the LSSmScarlet2 protein at a resolution of 1.41 Å. Site-directed mutagenesis of the LSSmScarlet2 protein demonstrated the key role of the T74 residue in improving the pH and chemical stability of the LSSmScarlet2 protein. Full article

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 (Gdm₂SO₄), 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 Gdm₂SO₄ at 25 °C. Lastly, we observed a novel non-monotonous temperature dependence of DNA origami denaturation in Gdm₂SO₄ 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. Full article

Halide perovskite solar cells (HPSCs) are promising photovoltaic materials due to their excellent optoelectronic properties, low cost, and high efficiency. Here, we demonstrate atmospheric solution processing and stability of cesium tin-lead triiodide (CsSnPbI₃) thin films for solar cell applications. The effect of additives, such as pyrazine and guanidinium thiocyanate (GuaSCN), on bandgap, film morphology, structure, and stability is investigated. Our results indicate the formation of a wide bandgap (>2 eV) structure with a mixed phase of tin oxide (SnO₂) and Cs(Sn, Pb)I₃. The addition of pyrazine decreases the intensity of SnO₂ peaks, but the bandgap does not change much. With the addition of GuaSCN, the bandgap of the films reduces to 1.5 eV, and a dendritic structure of Cs(Sn, Pb)I₃ is observed. GuaSCN addition also reduces the oxygen content in the films. To enable uniform film crystallization, cesium chloride (CsCl) and dimethyl sulfoxide (DMSO) additives are used in the precursor. Both CsCl and DMSO suppress dendrite formation with the latter resulting in uniform polycrystalline films with a bandgap of 1.5 eV. Heat and light soaking (HLS) stability tests at 65 °C and 1 sun for 100 h show all film types are stable with temperature but result in phase segregation with light exposure. Full article

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. Full article

Near-infrared fluorescent proteins (NIR FPs) based on the complexes of bacterial phytochromes with their natural biliverdin chromophore are widely used as genetically encoded optical probes for visualization of cellular processes and deep-tissue imaging of cells and organs in living animals. In this work, we show that the steady-state and kinetic dependencies of the various spectral characteristics of iRFP713, developed from the bacterial phytochrome RpBphP2 and recorded at protein unfolding induced by guanidine hydrochloride (GdnHCl), guanidine thiocyanate (GTC), and urea, differ substantially. A study of the unfolding of three single-tryptophan mutant forms of iRFP713 expectedly revealed that protein unfolding begins with the dissociation of the native dimer, while the monomers remain compact. A further increase in the denaturant concentration leads to the formation of an intermediate state of iRFP713 having hydrophobic areas exposed on the protein surface (I). The total surface charge of iRFP713 (pI 5.86) changes from negative to positive with an increase in the concentration of GdnHCl and GTC because the negative charge of glutamic and aspartic acids is neutralized by forming salt bridges between the carboxyl groups and GdnH⁺ ions and because the guanidinium cations bind to amide groups of glutamines and asparagines. The coincidence of both the concentration of the denaturants at which the intermediate state of iRFP713 accumulates and the concentration of GdnH⁺ ions at which the neutralization of the surface charge of the protein in this state is ensured results in strong protein aggregation. This is evidently realized by iRFP713 unfolding by GTC. At the unfolding of the protein by GdnHCl, an intermediate state is populated at higher denaturant concentrations and a strong aggregation is not observed. As expected, protein aggregates are not formed in the presence of the urea. The aggregation of the protein upon neutralization of the charge on the macromolecule surface is the main indicator of the intermediate state of protein. The unfolded state of iRFP713, whose formation is accompanied by a significant decrease in the parameter A, was found to have a different residual structure in the denaturants used. Full article