Protein Thermal Stability Research Articles

Cellular Thermal Shift Assay for the Detection of Small Molecule-Target Interactions in Arabidopsis Cells.

Chemical genetics takes advantage of small molecule-protein interactions to explore various biological processes. Although an attractive alternative to classical genetics in plants, the identification of small-molecule targets remains a challenge and limits the broad use of the compounds. The cellular thermal shift assay (CETSA), based on the principle that binding of small molecules could affect the thermal stability of proteins, has been applied for target validation in plant cells. Combined with high-resolution mass spectrometry, CETSA can detect small-molecule targets by monitoring the changes in the protein thermal stability caused by the interactions with small molecules at the proteome level. Here we describe the small-molecule target validation as well as the target identification with mass spectrometry by means of CETSA.

Dihydroflavonoid glycosides from Viscum album and their inhibitory effects on hepatic lipid accumulation and target identification

Five undescribed dihydroflavonoid glycoside derivatives, namely albvisosides A‒E, together with two known compounds were isolated from the roots and stem leaves of Viscum album L. var. album. (European mistletoe). Their structures were determined by HRESIMS, 1D and 2D NMR, and ECD analysis. Albvisoside B exhibits significant inhibitory effect on hepatic lipid accumulation in HepG2 cells at very low concentrations (EC50: 0.7 nM). Using proteome integral solubility alteration assay, the direct targets or downstream effectors of albvisoside B were elucidated. As a result, 97 proteins were identified based on ligand-induced alterations in the protein thermal stability. Bioinformatics analysis indicated that albvisoside B primarily ameliorated oleic acid-induced lipid accumulation by regulating the selenoamino acids metabolism signaling pathway. RPL3, ADAM17, and RPL14 were likely to be involved in mediating the lipid-lowering effect of albvisoside B.

Classically Synthesized ZIF-8 and Au/ZIF-8 for Biocompatibility Assessment with Hemoglobin

ZIF-8 and Au/ZIF-8 were synthesized and utilized to evaluate their biocompatibility with hemoglobin (Hb). Conformational changes were introduced by both materials in the native structure of Hb after interaction. Evidently, Au/ZIF-8 had greater structural influence than ZIF-8. The protein thermal stability was stabilized by both ZIF-8 and Au/ZIF-8 only at lower concentrations.

Integrated changes in thermal stability and proteome abundance during altered nutrient states in Escherichia coli and human cells.

Altered thermal solubility measurement techniques are emerging as powerful tools to assess ligand binding, post-translational modification, protein-protein interactions, and many other cellular processes that affect protein state under various cellular conditions. Thermal solubility or stability profiling techniques are enabled on a global proteomic scale by employing isobaric tagging reagents that facilitate multiplexing capacity required to measure changes in the proteome across thermal gradients. Key among these is thermal proteomic profiling (TPP), which requires 8-10 isobaric tags per gradient and generation of multiple proteomic datasets to measure different replicates and conditions. Furthermore, using TPP to measure protein thermal stability state across different conditions may also require measurements of differential protein abundance. Here, we use the proteome integral stability alteration (PISA) assay, a higher throughput version of TPP, to measure global changes in protein thermal stability normalized to their protein abundance. We explore the use of this approach to determine changes in protein state between logarithmic and stationary phase Escherichia coli as well as glucose-starved human Hek293T cells. We observed protein intensity-corrected PISA changes in 290 and 350 proteins due to stationary phase transition in E. coli and glucose starvation, respectively. These data reveal several examples of proteins that were not previously associated with nutrient states by abundance alone. These include E. coli proteins such as putative acyl-CoA dehydrogenase (aidB) and chaperedoxin (cnoX) as well as human RAB vesicle trafficking proteins and many others which may indicate their involvement in metabolic diseases such as cancer.

Conformational stability of ageritin, a metal binding ribotoxin-like protein of fungal origin

Ageritin is a ribotoxin-like protein of biotechnological interest, belonging to a family of ribonucleases from edible mushrooms. Its enzymatic activity is explicated through the hydrolysis of a single phosphodiester bond, located in the sarcin/ricin loop of ribosomes. Unlike other ribotoxins, ageritin activity requires divalent cations (Zn2+). Here we investigated the conformational stability of ageritin in the pH range 4.0–7.4, using calorimetric and spectroscopic techniques. We observed a high protein thermal stability at all pHs with a denaturation temperature of 78 °C. At pH 5.0 we calculated a value of 36 kJ mol−1 for the unfolding Gibbs energy at 25 °C. We also analysed the thermodynamic and catalytic behaviour of S-pyridylethylated form, obtained by alkylating the single Cys18 residue, which is predicted to bind Zn2+. We show that this form possesses the same activity and structure of ageritin, but lower stability. In fact, the corresponding values of 52 °C and 14 kJ mol−1 were found. Conservation of activity is consistent with the location of alkylation site on the opposite site of the catalytic site cleft. Inasmuch as Cys18 is part of a structurally stabilizing zinc-binding site, disrupted by cysteine alkylation, our results point to an important role of metal ions in ageritin stability.

Effect of Different Thawing Methods on the Physicochemical Properties and Microstructure of Frozen Instant Sea Cucumber.

To provide recommendations to users regarding which thawing method for frozen instant sea cucumbers entails lower quality losses, in this study we compared the water retention, mechanical properties, protein properties, and microstructures of frozen instant sea cucumbers post-thawing by means of different thawing approaches, including refrigerator thawing (RT), air thawing (AT), water immersion thawing (WT), and ultrasound-assisted thawing (UT). The results indicated that UT took the shortest time. RT samples exhibited the best water-holding capacity, hardness and rheological properties, followed by UT samples. The α-helix and surface hydrophobicity of WT and AT samples were significantly lower than those of the first two methods (p < 0.05). The lowest protein maximum denaturation temperature (Tmax) was obtained by means of WT. AT samples had the lowest maximum fluorescence emission wavelength (λmax). Based on these results, WT and AT were more prone to the degradation of protein thermal stability and the destruction of the protein structure. Similarly, more crimping and fractures of the samples after WT and AT were observed in the sea cucumbers’ microstructures. Overall, we observed that UT can be used to maintain the quality of frozen instant sea cucumbers in the shortest time.

Changes in protein oxidation, structure, and thermal stability of chicken breast subjected to ultrasound-assisted immersion freezing during frozen storage

The influence of ultrasound-assisted immersion freezing (UF), immersion freezing (IF), and air freezing (AF) on the protein oxidation, structure, and thermal stability of chicken breast during frozen storage was evaluated in this study. Compared to IF and AF samples, the UF samples had a lower carbonyl content, dityrosine content, and surface hydrophobicity of myofibrillar protein (MP) (P < 0.05), as well as a higher free amino group content and total and reactive sulfhydryl content (P < 0.05). Moreover, UF significantly delayed the deterioration of protein secondary and tertiary structures and the decrease in protein thermal stability during frozen storage (P < 0.05). Additionally, the UF samples at 180 days had similar protein structures and quality characteristics to the IF samples at 90 days or the AF samples at 60 days. Overall, UF treatment can effectively retard protein oxidation, protein structure deterioration, and protein thermal stability loss caused by frozen storage.

Spatial organization of hydrophobic and charged residues affects protein thermal stability and binding affinity

What are the molecular determinants of protein–protein binding affinity and whether they are similar to those regulating fold stability are two major questions of molecular biology, whose answers bring important implications both from a theoretical and applicative point of view. Here, we analyze chemical and physical features on a large dataset of protein–protein complexes with reliable experimental binding affinity data and compare them with a set of monomeric proteins for which melting temperature data was available. In particular, we probed the spatial organization of protein (1) intramolecular and intermolecular interaction energies among residues, (2) amino acidic composition, and (3) their hydropathy features. Analyzing the interaction energies, we found that strong Coulombic interactions are preferentially associated with a high protein thermal stability, while strong intermolecular van der Waals energies correlate with stronger protein–protein binding affinity. Statistical analysis of amino acids abundances, exposed to the molecular surface and/or in interaction with the molecular partner, confirmed that hydrophobic residues present on the protein surfaces are preferentially located in the binding regions, while charged residues behave oppositely. Leveraging on the important role of van der Waals interface interactions in binding affinity, we focused on the molecular surfaces in the binding regions and evaluated their shape complementarity, decomposing the molecular patches in the 2D Zernike basis. For the first time, we quantified the correlation between local shape complementarity and binding affinity via the Zernike formalism. In addition, considering the solvent interactions via the residue hydropathy, we found that the hydrophobicity of the binding regions dictates their shape complementary as much as the correlation between van der Waals energy and binding affinity. In turn, these relationships pave the way to the fast and accurate prediction and design of optimal binding regions as the 2D Zernike formalism allows a rapid and superposition-free comparison between possible binding surfaces.

Proteome-wide analysis of stress response to temperature in Sulfolobus islandicus

Sulfolobus islandicus is thermophilic archaea that live in an extreme environment of 75 °C–80 °C and pH 2–3. Currently, the molecular mechanism of archaeal adaptation to high temperatures and the stability of proteins at high temperatures are still unclear. This study utilizes proteomics to analyze the differential expression of S. islandicus proteins at different temperatures. We found that ribosomes, glycolysis, nucleotide metabolism, RNA metabolism, transport system, and sulfur metabolism are all affected by temperature. Methylation modification of some proteins changed with temperature. Thermal proteome profiling (TPP) was used to analyze the thermal stability of proteins under 65 °C–85 °C growth conditions. It is suggested that the Tm values of proteins are mainly distributed around the optimum growth temperature (OGT). The proteins in the glycolysis pathway had high thermal stability. Meanwhile, proteins related to DNA replication and translation showed low thermal stability. The protein thermal stability of S. islandicus cultured under 65 °C and 85 °C was higher than that of 75 °C. Our study reveals that S. islandicus may adapt to temperature changes by regulating protein synthesis and carbon metabolism pathways, changing post-translational modifications, and improving protein stability at the same time. SignificanceThe molecular mechanism of archaeal adaptation to high temperatures and the stability of proteins at high temperatures are still unclear. Our proteomics study identified 477 differentially expressed proteins of S. islandicus at different temperatures, suggesting that ribosomes, glycolysis, nucleotide metabolism, RNA metabolism, transport system, and sulfur metabolism are affected by temperature. Meanwhile, we found that methylation modification of some proteins changed with temperature. To evaluate the thermal stability of the proteome, we performed thermal proteome profiling to analyze the Tm of proteins under 65 °C–85 °C growth conditions. Tm values of proteins are mainly distributed around the optimum growth temperature. The proteins in the glycolysis pathway had high thermal stability. Meanwhile, proteins related to DNA replication and translation showed low thermal stability. Our study reveals that S. islandicus may adapt to temperature changes by regulating protein synthesis and carbon metabolism pathways, changing post-translational modifications, and improving protein stability at the same time.

The Hidden Intricacies of Aquaporins: Remarkable Details in a Common Structural Scaffold.

Evolution turned aquaporins (AQPs) into the most efficient facilitators of passive water flow through cell membranes at no expense of solute discrimination. In spite of a plethora of solved AQP structures, many structural details remain hidden. Here, by combining extensive sequence- and structural-based analysis of a unique set of 20 non-redundant high-resolution structures and molecular dynamics simulations of four representatives, key aspects of AQP stability, gating, selectivity, pore geometry, and oligomerization, with a potential impact on channel functionality, are identified. The general view of AQPs possessing a continuous open water pore is challenged and it is depicted that AQPs' selectivity is not exclusively shaped by pore-lining residues but also by the relative arrangement of transmembrane helices. Moreover, this analysis reveals that hydrophobic interactions constitute the main determinant of protein thermal stability. Finally, a numbering scheme of the conserved AQP scaffold is established, facilitating direct comparison of, for example, disease-causing mutations and prediction of potential structural consequences. Additionally, the results pave the way for the design of optimized AQP water channels to be utilized in biotechnological applications.

Thermal Shift Assay for Small GTPase Stability Screening: Evaluation and Suitability.

Thermal unfolding methods are commonly used as a predictive technique by tracking the protein’s physical properties. Inherent protein thermal stability and unfolding profiles of biotherapeutics can help to screen or study potential drugs and to find stabilizing or destabilizing conditions. Differential scanning calorimetry (DSC) is a ‘Gold Standard’ for thermal stability assays (TSA), but there are also a multitude of other methodologies, such as differential scanning fluorimetry (DSF). The use of an external probe increases the assay throughput, making it more suitable for screening studies, but the current methodologies suffer from relatively low sensitivity. While DSF is an effective tool for screening, interpretation and comparison of the results is often complicated. To overcome these challenges, we compared three thermal stability probes in small GTPase stability studies: SYPRO Orange, 8-anilino-1-naphthalenesulfonic acid (ANS), and the Protein-Probe. We studied mainly KRAS, as a proof of principle to obtain biochemical knowledge through TSA profiles. We showed that the Protein-Probe can work at lower concentration than the other dyes, and its sensitivity enables effective studies with non-covalent and covalent drugs at the nanomolar level. Using examples, we describe the parameters, which must be taken into account when characterizing the effect of drug candidates, of both small molecules and Designed Ankyrin Repeat Proteins.

N-linked glycosylation enhances hemagglutinin stability in avian H5N6 influenza virus to promote adaptation in mammals.

Clade 2.3.4.4 avian H5Ny viruses, namely H5N2, H5N6, and H5N8, have exhibited unprecedented intercontinental spread in poultry. Among them, only H5N6 viruses are frequently reported to infect mammals and cause serious human infections. In this study, the genetic and biological characteristics of surface hemagglutinin (HA) from clade 2.3.4.4 H5Ny avian influenza viruses (AIVs) were examined for adaptation in mammalian infection. Phylogenetic analysis identified an amino acid (AA) deletion at position 131 of HA as a distinctive feature of H5N6 virus isolated from human patients. This single AA deletion was found to enhance H5N6 virus replication and pathogenicity in vitro and in mammalian hosts (mice and ferrets) through HA protein acid and thermal stabilization that resulted in reduced pH threshold from pH 5.7 to 5.5 for viral-endosomal membrane fusion. Mass spectrometry and crystal structure revealed that the AA deletion in HA at position 131 introduced an N-linked glycosylation site at 129, which increases compactness between HA monomers, thus stabilizes the trimeric structure. Our findings provide a molecular understanding of how HA protein stabilization promotes cross-species avian H5N6 virus infection to mammalian hosts.

Role of ionic liquids on stabilization of therapeutic proteins and model proteins.

Ionic liquids (ILs) exhibit potential as excipients to stabilize proteins in solutions. This mini-review is not a detailed reference book on ILs, rather a brief overview of the main achievements published in the literature on their effect on protein aggregation, unfolding, structural and thermal stability, and activity. The main focus of the manuscript is three widely studied groups of ionic liquids: imidazolium-, cholinium- and alkylammonium-based and their effect on the model and therapeutic proteins.

Microtiter Plate-Based Differential Scanning Fluorimetry: A High-Throughput Method for Efficient Formulation Development

Nano/microparticles are widely used as vaccine adjuvants to improve antigen stability and enhance immune response. Conformational stability of a given protein was normally assessed using differential scanning calorimetry (DSC) for the optimization of formulation and for ensuring antigen stability in vaccine products. Here, a higher throughput version, namely the microtiter plate-based differential scanning fluorimetry (DSF) method was developed and optimized for assessing the protein thermal stability in the particulate adjuvant-adsorbed form. Using recombinant human papillomavirus (HPV) vaccine antigens, along with several model proteins, enhanced sensitivity and correlation to the well-established differential scanning calorimetry were demonstrated. Higher throughput and much smaller sample consumption (1/10 ∼ 1/20 of the amount needed as compared to DSC) make the plate-based DSF a method of choice for formulation development, particularly during the early developmental phase of a project where the sample amount is usually quite limited.

Lentil Protein and Tannic Acid Interaction Limits in Vitro Peptic Hydrolysis and Alters Peptidomic Profiles of the Proteins.

In this study, the nature of lentil protein-tannic acid (LPTA) interaction and its effect on in vitro pepsin digestion were investigated. LPTA mixtures containing 1% w/v LP and 0.001-0.5% TA were prepared and characterized in terms of particle size, thermal properties, and secondary and tertiary structures. A 20-fold increase in particle size was observed in LPTA0.5% compared to LP control (without TA), indicating aggregation. Static quenching of tryptophan residues within the protein hydrophobic folds was observed. Increasing TA levels also enhanced protein thermal stability. Over 50% reduction in free amino groups of LPTA 0.5%, relative to LP, was observed after pepsin digestion. Cleavage specificity of pepsin and peptidomic profile of LP were modified by the presence of TA in LPTA 0.5%. This study showed that 0.5% w/v TA induced protein aggregation and reduced LP digestibility by hindering the accessibility of pepsin to the protein network, thus modifying the profile of released peptides.

Unique structural and dynamic properties of the HNH nuclease in mesophilic and thermophilic Cas9 revealed by NMR

CRISPR‐Cas9 is a widely utilized biochemical tool with applications in biotechnology and in medicine. Target DNA recognition and nuclease sites of Cas9 are spatially separated yet functionally coupled, indicative of an allosteric crosstalk throughout the enzyme. There are structural and dynamic changes occuring throughout Cas9 that are critical for this signal propagation. However, an understanding of the molecular motions guiding Cas9 and how this influences its mechanism are unknown. In this study we report on the structural and dynamic properties of the HNH domain between the extensively studied SpCas9 from Streptococcus pyogenes, and a recently discovered thermostable Cas9 homolog from a thermophilic bacterium Geobacillus stearothermophilus(GeoCas9). Here we show that despite sequential and structural conservation, SpHNH and GeoHNH have different dynamic profiles. We found the intrinsic dynamics of SpHNH to be on the slow (ms) timescale, while GeoHNH is highly influenced by fast (ps‐ns) timescale dynamics. Furthermore, we found that when the residue with the highest flexibility on the ps‐ns timescale is mutated in GeoHNH, its protein solubility, thermal stability, and dynamic profile are all drastically distorted. Our results demonstrate how there are mechanistic differences between the HNH domain of mesophilic and thermophilic Cas9 species. Our results also highlight how these fast timescale motions that regulate GeoHNH play a role in giving GeoCas9 its thermophilic characteristic.

Supramolecular Assembly of His-Tagged Fluorescent Protein Guests within Coiled-Coil Peptide Crystal Hosts: Three-Dimensional Ordering and Protein Thermal Stability.

The use of biomaterials for the inclusion and stabilization of biopolymers is an ongoing challenge. Herein, we disclose three-dimensional (3D) coiled-coil peptide crystals with metal ions that include and overgrow His-tagged fluorescent proteins within the crystal. The protein guests are found within two symmetry-related growth sectors of the crystalline host that are associated with faces of the growing crystal that display ligands for metal ions. The fluorescent proteins are included within this "hourglass" region of the crystals at a notably high level, display order within the crystal hosts, and demonstrate sufficiently tight packing to enable energy transfer between a donor-acceptor pair. His-tagged fluorescent proteins display remarkable thermal stability to denaturation over extended periods of time (days) at high temperatures when within the crystals. Ultimately, this strategy may prove useful for the prolonged storage of thermally sensitive biopolymer guests within a 3D crystalline matrix.

INSIGHT INTO THE THERMOSTABILITY OF NOVEL FLUORESCENT PROTEIN ISOLATED FROM SEA ANEMONE CRIBRINOPSIS JAPONICA: IN SILICO STUDY

Fluorescent protein has been applied in various diagnostic and biotechnology application. However, in these applications, the temperature conditions are simulated at higher temperatures of 289K and 300K compared to natural sea anemone Cribrinopsis japonica fluorescent protein. This study evaluates the predictive structure and the molecular dynamic interaction of the protein with its target in different external temperature using ab initio similarity modeling and GROMACS/VMD respectively. Three-dimensional structure of the protein, named cjFP510 were predicted and analysed based on the highest similarity template model of Anemonia sulcata (2c9i) at 66.97%. The predicted model shows alternating a and β helices with longer loops and extra a-helix. cjFP510 was programmed with molecular dynamic simulation with template protein 2c9i as a reference to study its comparative adaptability in two different temperatures of 289K and 300K. cjFP510 was found to be more stable at both temperatures compared to 2c9i. Further simulation was conducted on the gyration radius to evaluate the compactness of the protein folding. Lower gyration radius of cjFP510 denotes more stable protein at 289K simulated environment than 29ci. This may be due to the presence of an extra a -helix based on the predicted model and few amino acid residues such as glycine, lysine, and arginine which contributed to the protein flexibility and thermal stability Conclusively, cjFP510 is more thermostable in the two conditional temperatures tested.

Bovine serum albumin with gallic acid: Molecular modeling and physicochemical profiling

Introduction. Gallic acid is a biologically active natural compound with strong antioxidant properties. Gallic acid is highly soluble and stable. It is known to increase the thermal stability of protein. However, its bioavailability is low, but interaction with proteins can solve this problem. Bovine serum albumin can bind various ligands, including polyphenols. The resulting complex of gallic acid and bovine serum albumin can become a promising functional food additive.&#x0D; Study objects and methods. This research featured in silico molecular modeling of gallic acid and bovine serum albumin using the HyperChem program. The methods of infrared spectrometry, potentiometry, and sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) made it possible to describe the physicochemical profile of the complex.&#x0D; Results and discussion. The molecular modeling confirmed that hydrophobic interactions were responsible for the chemical bond between gallic acid and bovine serum albumin. The SDS-PAGE test showed that the protein molecule remained intact. The reducing properties of the complex grew as the concentration of gallic acid increased. At 100 mg/L of gallic acid, the reducing properties were 7.8 ± 1.3 mg/L equivalent of gallic acid. At 200 and 300 mg/L, the values reached 15.90 ± 2.65 and 23.30 ± 5.05 mg/L, respectively. The IR spectrometry revealed a significant difference between the samples with different concentrations of gallic acid.&#x0D; Conclusion. The research managed to predict the properties of the complex of bovine serum albumin and gallic acid during its formation. The resulting complex had the highest reducing properties at 0.69 g of bovine serum albumin and 300 mg of gallic acid. The obtained parameters can be used in the food industry to develop new food additives.

Structure and activity of a thermally stable mutant of Acanthamoeba actophorin.

Actophorin, which was recently tested for crystallization under microgravity on the International Space Station, was subjected to mutagenesis to identify a construct with improved biophysical properties that were expected to improve the extent of diffraction. First, 20 mutations, including one C-terminal deletion of three residues, were introduced individually into actophorin, resulting in modest increases in thermal stability of between +0.5°C and +2.2°C. All but two of the stabilizing mutants increased both the rates of severing F-actin filaments and of spontaneous polymerization of pyrenyl G-actin in vitro. When the individual mutations were combined into a single actophorin variant, Acto-2, the overall thermal stability was 22°C higher than that of wild-type actophorin. When an inactivating S2P mutation in Acto-2 was restored, Acto-2/P2S was more stable by 20°C but was notably more active than the wild-type protein. The inactivating S2P mutation reaffirms the importance that Ser2 plays in the F-actin-severing reaction. The crystal structure of Acto-2 was solved to 1.7 Å resolution in a monoclinic space group, a first for actophorin. Surprisingly, despite the increase in thermal stability, the extended β-turn region, which is intimately involved in interactions with F-actin, is disordered in one copy of Acto-2 in the asymmetric unit. These observations emphasize the complex interplay among protein thermal stability, function and dynamics.