Thermal Denaturation Analysis Research Articles

Synthesis and properties of RNA constrained by a 2'-O-disulfide bridge.

We recently reported the properties of RNA hairpins constrained by a dimethylene (DME) disulfide (S-S) linker incorporated between two adjacent nucleosides in the loop and showed that this linker locked the hairpin conformation thus disturbing the duplex/hairpin equilibrium. We have now investigated the influence of the length of the linker and synthesized oligoribonucleotides containing diethylene (DEE) and dipropylene (DPE) S-S bridges. This was achieved via the preparation of building blocks, namely 2'-O-acetylthioethyl (2'-O-AcSE) and 2'-O-acetylthiopropyl (2'-O-AcSP) uridine phosphoramidites, which were successfully incorporated into RNA sequences. Thermal denaturation analysis revealed that the DEE and DPE disulfide bridges destabilize RNA duplexes but do not disrupt the hairpin conformation. Furthermore, our investigation of the duplex/hairpin equilibrium indicated that sequences modified with DME and DEE S-S linkers predominantly lock the hairpin form, whereas the DPE S-S linker provides flexibility. These findings highlight the potential of S-S linkers to study RNA interactions.

Binding to the conserved and stably folded guide RNA pseudoknot induces Cas12a conformational changes during ribonucleoprotein assembly

Ribonucleoproteins (RNPs) comprise one or more RNA and protein molecules that interact to form a stable complex, which commonly involves conformational changes in the more flexible RNA components. Here, we propose that Cas12a RNP assembly with its cognate CRISPR RNA (crRNA) guide instead proceeds primarily through Cas12a conformational changes during binding to more stable, prefolded crRNA 5' pseudoknot handles. Phylogenetic reconstructions and sequence and structure alignments revealed that the Cas12a proteins are divergent in sequence and structure while the crRNA 5' repeat region, which folds into a pseudoknot and anchors binding to Cas12a, is highly conserved. Molecular dynamics simulations of three Cas12a proteins and their cognate guides revealed substantial flexibility for unbound apo-Cas12a. In contrast, crRNA 5' pseudoknots were predicted to be stable and independently folded. Limited trypsin hydrolysis, differential scanning fluorimetry, thermal denaturation, and CD analyses supported conformational changes of Cas12a during RNP assembly and an independently folded crRNA 5' pseudoknot. This RNP assembly mechanism may be rationalized by evolutionary pressure to conserve CRISPR loci repeat sequence, and therefore guide RNA structure, to maintain function across all phases of the CRISPR defense mechanism.

Loss of stability and unfolding cooperativity in hPGK1 upon gradual structural perturbation of its N-terminal domain hydrophobic core

Phosphoglycerate kinase has been a model for the stability, folding cooperativity and catalysis of a two-domain protein. The human isoform 1 (hPGK1) is associated with cancer development and rare genetic diseases that affect several of its features. To investigate how mutations affect hPGK1 folding landscape and interaction networks, we have introduced mutations at a buried site in the N-terminal domain (F25 mutants) that either created cavities (F25L, F25V, F25A), enhanced conformational entropy (F25G) or introduced structural strain (F25W) and evaluated their effects using biophysical experimental and theoretical methods. All F25 mutants folded well, but showed reduced unfolding cooperativity, kinetic stability and altered activation energetics according to the results from thermal and chemical denaturation analyses. These alterations correlated well with the structural perturbation caused by mutations in the N-terminal domain and the destabilization caused in the interdomain interface as revealed by H/D exchange under native conditions. Importantly, experimental and theoretical analyses showed that these effects are significant even when the perturbation is mild and local. Our approach will be useful to establish the molecular basis of hPGK1 genotype–phenotype correlations due to phosphorylation events and single amino acid substitutions associated with disease.

A comparative assessment of collagen type 1 from silver carp (fresh water) and milk shark(marine) fish waste.

In this study, a comparative structural and bioactive analysis of collagen extracted from the skin of bony and cartilaginous fishes. The acid-soluble method was followed to extract the collagen from Hypophthalmichthys molitrix (silver carp) andRhizoprionodon acutus (milk shark) followed by purification using Ion exchange chromatography. A higher yield of collagen was obtained from the skin of SCsk (69.45%) as compared to SHsk (55.29%). SDS PAGE displayed the characteristic α, β bands of the collagen type1. The native conformation and secondary structure stability of collagen were confirmed by FTIR, XRD and CD studies. The SEM micrographs exhibited the layered and fibrillar nature of the collagen from SHsk and SCsk, respectively. Relative solubility and thermal denaturation analysis showed SCsk to be more stable, but SHsk could withstand higher temperatures. 53.65% of antioxidant activity was observed in SCsk collagen as compared to SHsk (45.9%). Haemocompatibility, cell viability and adhesion results also displayed silvercarp skin to be a better source than Shark skin collagen. The results establish the potential of silver carp collagen as a biomaterial that can have many commercial applications in tissue engineering, cosmetics and food industries.

Equivalence of the transition heat capacities of proteins and DNA

It has been reported for many globular proteins that the native heat capacity at 25 °C, per gram, is the same. This has been interpreted to indicate that heat capacity is a fundamental property of native proteins that provides important information on molecular structure and stability. Heat capacities for both proteins and DNA has been suggested to be related to universal effects of hydration/solvation on native structures. Here we report on results from thermal denaturation analysis of two well-known proteins, human serum albumin and lysozyme, and a short DNA hairpin. The transition heat capacities at the Tm for the three molecules were quantitatively evaluated by differential scanning calorimetry. When normalized per gram rather than per mol the transition heat capacities were found to be precisely equivalent. This observation for the transition heat capacities of the proteins is consistent with previous reports. However, an identical transition heat capacity for DNA has not been reported and was unexpected. Further analysis of the collected data suggested a mass dependence of hydration effects on thermal denaturation that is preserved at the individual protein amino acid and DNA base levels. Equivalence of transition heat capacities suggests the possibility of a universal role of hydration effects on the thermal stability of both proteins and DNA.

Scleral growth stunting via sub-Tenon injection of cross-linking solutions in live rabbits

BackgroundScleral cross-linking is a potential method to inhibit axial elongation of the eye, preventing the progression of pathological myopia. Formaldehyde releasers, which are common preservatives found in cosmetics and ophthalmic...

Rosmarinic acid inhibits DNA glycation and modulates the expression of Akt1 and Akt3 partially in the hippocampus of diabetic rats

Non-enzymatic glycation of DNA and the associated effects are among pathogenic factors in diabetes mellitus. Natural polyphenols have anti-diabetic activity. Herein, the protective role of one of the phytochemicals, rosmarinic acid (RA), was evaluated in glycation (with fructose) of human DNA and expression of Akt genes in the hippocampus of diabetic rats. In-vitro studies using fluorescence, agarose gel electrophoresis, fluorescence microscopy, and thermal denaturation analyses revealed that glycation causes DNA damage and that RA inhibits it. In-vivo studies were performed by induction of diabetes in rats using streptozotocin. The diabetic rats were given RA daily through gavage feeding. The expression of Akt genes (inhibitors of apoptosis) in the hippocampus was evaluated using RT-qPCR. In diabetic rats, Akt1 and Akt3 were significantly down-regulated compared to the control group. Treating the diabetic rats with RA returned the expression of Akt1 and Akt3 relatively to the normal condition. Past studies have shown that diabetes induces apoptosis in the hippocampal neurons. Given that glycation changes the genes expression and causes cell death, apoptosis of the hippocampal neurons can be due to the glycation of DNA. The results also suggest that RA has reliable potency against the gross modification of DNA under hyperglycemic conditions.

Ligand binding constants for human serum albumin evaluated by ratiometric analysis of DSC thermograms

This paper describes an expanded application of our recently reported method (Eskew et al., Analytical Biochemistry 621,1 2021) utilizing thermogram signals for thermal denaturation measured by differential scanning calorimetry. Characteristic signals were used to quantitatively evaluate ligand binding constants for human serum albumin. In our approach the ensemble of temperature dependent calorimetric responses for various protein-ligand mixtures and native HSA were compared, in a ratiometric manner, to extract binding constants and stoichiometries. Protein/ligand mixtures were prepared at various ligand concentrations and subjected to thermal denaturation analysis by calorimetry. Measurements provided the melting temperature, Tm, and free-energy ΔGcal(37°C) for melting ligand-bound Albumin as a function of ligand concentration. Concentration dependent behaviors of these parameters derived from protein/ligand mixtures were used to construct dose-response curves. Fitting of dose-response curves yielded quantitative evaluation of the ligand binding constant and semi-quantitative estimates of the binding stoichiometry. Many of the ligands had known binding affinity for Albumin with binding constants reported in the literature. Evaluated binding parameters for the ligands impressively agreed with reported literature values determined using other standard experimental methods. Results are reported for 29 drug ligands binding to Albumin. These validate our calorimetry-based process for applications in pre-clinical drug screening.

Characterization of In Vitro G-Quadruplex Formation of Imetelstat Telomerase Inhibitor

Imetelstat (GRN163L) is a potent and specific telomerase inhibitor currently in clinical development for the treatment of hematological malignancies such as myelofibrosis and myelodysplastic syndrome. It is a 13-mer N3'-P5' thio-phosphoramidate oligonucleotide covalently functionalized at the 5'-end with a palmitoyl lipid moiety through an aminoglycerol linker. As a competitive inhibitor of human telomerase, imetelstat directly binds to the telomerase RNA component sequence (hTR) in the catalytic site of the enzyme and acts as a direct competitor of human telomere binding. Administration of imetelstat causes progressive shortening of the telomeres, thereby inhibiting malignant cells' proliferation. We report here the ability of imetelstat to form stable, parallel, intermolecular G-quadruplex structures in vitro. The impact of the ionic environment on the formation and stability of imetelstat higher-order structure was investigated through circular dichroism spectroscopy, thermal denaturation analysis, and size-exclusion chromatography. We demonstrated that different structural elements, such as the 5'-palmitoyl linker and the thio-phosphoramidate backbone, critically contribute to G-quadruplex stability. Experiments further showed that G-quadruplex formation does not hamper binding to the hTR oligonucleotide sequence in vitro.

Synthesis and investigation of quadruplex-DNA-binding, 9-O-substituted berberine derivatives.

A small series of five novel berberine derivatives was synthesized by the Cu-catalyzed click reaction of 9-propargyladenine with 9-O-(azidoalkyl)berberine derivatives. The association of the resulting berberine–adenine conjugates with representative quadruplex-forming oligonucleotides 22AG dA(G3TTA)3G3 and a2 d(ACAG4TGTG4)2 was examined with photometric and fluorimetric titrations, thermal DNA denaturation analysis, and CD spectroscopy. The results from the spectrometric titrations indicated the formation of 2:1 or 1:1 complexes (ligand:G4-DNA) with log Kb values of 10–11 (2:1) and 5–6 (1:1), which are typical for berberine derivatives. Notably, a clear relationship between the binding affinity of the ligands with the length of the alkyl linker chain, n, was not observed. However, depending on the structure, the ligands exhibited different effects when bound to the G4-DNA, such as fluorescent light-up effects and formation of ICD bands, which are mostly pronounced with a linker length of n = 4 (with a2) and n = 5 (with 22AG), thus indicating that each ligand–G4-DNA complex has a specific structure with respect to relative alignment and conformational flexibility of the ligand in the binding site. It was shown exemplarily with one representative ligand from the series that such berberine–adenine conjugates exhibit a selective binding, specifically a selectivity to quadruplex DNA in competition with duplex DNA, and a preferential thermal stabilization of the G4-DNA forms 22AG and KRAS. Notably, the experimental data do not provide evidence for a significant effect of the adenine unit on the binding affinity of the ligands, for example, by additional association with the loops, presumably because the adenine residue is sterically shielded by the neighboring triazole unit.

Structurally plastic NEMO and oligomerization prone IKK2 subunits define the behavior of human IKK2:NEMO complexes in solution

The human IκB Kinase (IKK) is a multisubunit protein complex of two kinases and one scaffolding subunit that controls induction of transcription factor NF-κB activity. IKK behaves as an entity of aberrantly high apparent molecular weight in solution. Recent X-ray crystallographic and cryo-electron microscopy structures of individual catalytic subunits (IKK1/IKKα and IKK2/IKKβ) reveal that they are both stably folded dimeric proteins that engage in extensive homo-oligomerization through unique surfaces that are required for activation of their respective catalytic activities. The NEMO/IKKγ subunit is a predominantly coiled coil protein that is required for activation of IKK through the canonical NF-κB signaling pathway. Here we report size-exclusion chromatography, multi-angle light scattering, analytical centrifugation, and thermal denaturation analyses of full-length human recombinant NEMO as well as deletion and disease-linked variants. We observe that NEMO is predominantly a dimer in solution, although by virtue of its modular coiled coil regions NEMO exhibits complicated solution dynamics involving portions that are mutually antagonistic toward homodimerization. This behavior causes NEMO to behave as a significantly larger sized particle in solution. Analyses of NEMO in complex with IKK2 indicate that NEMO preserves this structurally dynamic character within the multisubuit complex and provides the complex-bound IKK2 further propensity toward homo-oligomerization. These observations provide critical information on the structural plasticity of NEMO subunit dimers which helps clarify its role in diseases and in IKK regulation through oligomerization-dependent phosphorylation of catalytic IKK2 subunit dimers.

Improved Production and Biophysical Analysis of Recombinant Silicatein-α.

Silicatein-α is a hydrolase found in siliceous sea sponges with a unique ability to condense and hydrolyse silicon–oxygen bonds. The enzyme is thus of interest from the perspective of its unusual enzymology, and for potential applications in the sustainable synthesis of siloxane-containing compounds. However, research into this enzyme has previously been hindered by the tendency of silicatein-α towards aggregation and insolubility. Herein, we report the development of an improved method for the production of a trigger factor-silicatein fusion protein by switching the previous hexahistidine tag for a Strep-II tag, resulting in 244-fold improvement in protein yield compared to previous methods. Light scattering and thermal denaturation analyses show that under the best storage conditions, although oligomerisation is never entirely abolished, these nanoscale aggregates of the Strep-tagged protein exhibit improved colloidal stability and solubility. Enzymatic assays show that the Strep-tagged protein retains catalytic competency, but exhibits lower activity compared to the His6-tagged protein. These results suggest that the hexahistidine tag is capable of non-specific catalysis through their imidazole side chains, highlighting the importance of careful consideration when selecting a purification tag. Overall, the Strep-tagged fusion protein reported here can be produced to a higher yield, exhibits greater stability, and allows the native catalytic properties of this protein to be assessed.

Evidence of Destabilization of the Human Thymidylate Synthase (hTS) Dimeric Structure Induced by the Interface Mutation Q62R.

In human cells, thymidylate synthase (TS) provides the only source of 2′-deoxythymidyne-5′-monophosphate (dTMP), which is required for DNA biosynthesis. Because of its pivotal role, human TS (hTS) represents a validated target for anticancer chemotherapy. Nonetheless, the efficacy of drugs blocking the hTS active site has limitations due to the onset of resistance in cancer cells, requiring the identification of new strategies to effectively inhibit this enzyme. Human TS works as an obligate homodimer, making the inter-subunit interface an attractive targetable area. Here, we report the design and investigation of a new hTS variant, in which Gln62, located at the dimer interface, has been replaced by arginine in order to destabilize the enzyme quaternary assembly. The hTS Q62R variant has been characterized though kinetic assay, thermal denaturation analysis and X-ray crystallography. Our results provide evidence that hTS Q62R has a reduced melting temperature. The effective destabilization of the TS quaternary structure is also confirmed by structural analysis, showing that the introduced mutation induces a slight aperture of the hTS dimer. The generation of hTS variants having a more accessible interface area can facilitate the screening of interface-targeting molecules, providing key information for the rational design of innovative hTS interface inhibitors.

HgII binds to C-T mismatches with high affinity.

Binding reactions of HgII and AgI to pyrimidine-pyrimidine mismatches in duplex DNA were characterized using fluorescent nucleobase analogs, thermal denaturation and 1H NMR. Unlike AgI, HgII exhibited stoichiometric, site-specific binding of C–T mismatches. The on- and off-rates of HgII binding were approximately 10-fold faster to C–T mismatches (kon ≈ 105 M−1 s−1, koff ≈ 10−3 s−1) as compared to T–T mismatches (kon ≈ 104 M−1 s−1, koff ≈ 10−4 s−1), resulting in very similar equilibrium binding affinities for both types of ‘all natural’ metallo base pairs (Kd ≈ 10–150 nM). These results are in contrast to thermal denaturation analyses, where duplexes containing T–T mismatches exhibited much larger increases in thermal stability upon addition of HgII (ΔTm = 6–19°C), as compared to those containing C–T mismatches (ΔTm = 1–4°C). In addition to revealing the high thermodynamic and kinetic stabilities of C–HgII–T base pairs, our results demonstrate that fluorescent nucleobase analogs enable highly sensitive detection and characterization of metal-mediated base pairs – even in situations where metal binding has little or no impact on the thermal stability of the duplex.

Insights into the molecular interaction between sucrose and α-chymotrypsin

One of the most important purposes of enzyme engineering is to increase the thermal and kinetic stability of enzymes, which is an important factor for using enzymes in industry. The purpose of the present study is to achieve a higher thermal stability of α-chymotrypsin (α-Chy) by modification of the solvent environment. The influence of sucrose was investigated using thermal denaturation analysis, fluorescence spectroscopy, circular dichroism, molecular docking and molecular dynamics (MD) simulations. The results point to the effect of sucrose in enhancing the α-Chy stability. Fluorescence spectroscopy revealed one binding site that is dominated by static quenching. Molecular docking and MD simulation results indicate that hydrogen bonding and van der Waals forces play a major role in stabilizing the complex. Tm of this complex was enhanced due to the higher H-bond formation and the lower surface hydrophobicity after sucrose modification. The results show the ability of sucrose in protecting the native structural conformation of α-Chy. Sucrose was preferentially excluded from the surface of α-Chy which is explained by the higher tendency of water toward favorable interactions with the functional groups of α-Chy than with sucrose.

PH-Dependent Switching of Base Pairs Using Artificial Nucleobases with Carboxyl Groups.

In this study, we report the synthesis of modified oligonucleotides consisting of benzoic acid or isophthalic acid residues as new nucleobases. As evaluated by UV thermal denaturation analysis at different pH conditions (5.0, 6.0, 7.0, and 8.0), these modified oligonucleotides exhibited pH-dependent recognition of natural nucleobases and one is first found to be capable of base pair switching in response to a pH change. The isophthalic acid residue incorporated into the oligonucleotide on a d-threoninol backbone could preferentially bind with adenine but with guanine in response to a change in the pH conditions from pH 5 to pH 7 (or 8) without significant difference in duplex stability. These findings would be valuable for further developing pH-responsive DNA-based molecular devices.

Three new mixed‐ligand copper(II) complexes containing glycyl‐l‐valine and N,N‐aromatic heterocyclic compounds: Synthesis, characterization, DNA interaction, cytotoxicity and antimicrobial activity

Three novel copper(II) complexes, [Cu(Gly‐l‐Val)(HPBM)(H2O)]·ClO4·H2O (1), [Cu(Gly‐l‐Val)(TBZ)(H2O)]·ClO4 (2) and [Cu(Gly‐l‐Val)(PBO)(H2O)]·ClO4 (3) (Gly‐l‐Val = glycyl‐l‐valine anion, HPBM = 5‐methyl‐2‐(2′‐pyridyl)benzimidazole, TBZ = 2‐(4′‐thiazolyl)benzimidazole, PBO = 2‐(2′‐pyridyl)benzoxazole), have been prepared and characterized with elemental analyses, conductivity measurements as well as various spectroscopic techniques. The interactions of these copper complexes with calf thymus DNA were explored using UV–visible, fluorescence, circular dichroism, thermal denaturation, viscosity and docking analyses methods. The experimental results showed that all three complexes could bind to DNA via an intercalative mode. Moreover, the cytotoxic effects were evaluated using the MTT method, and the antimicrobial activity of these complexes was tested against Bacillus subtilis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa. The results showed that the activities are consistent with their DNA binding abilities, following the order of 1 > 2 > 3.

P190RhoGAP proteins contain pseudoGTPase domains

The two p190RhoGAP proteins, p190RhoGAP-A and -B, are key regulators of Rho GTPase signaling and are essential for actin cytoskeletal structure and contractility. Here we report the discovery of two evolutionarily conserved GTPase-like domains located in the ‘middle domain’, previously thought to be unstructured. Deletion of these domains reduces RhoGAP activity. Crystal structures, MANT-GTPγS binding, thermal denaturation, biochemical assays and sequence homology analysis all strongly support defects in nucleotide-binding activity. Analysis of p190RhoGAP proteins therefore indicates the presence of two previously unidentified domains which represent an emerging group of pseudoenzymes, the pseudoGTPases.

Engineered holocytochrome c synthases that biosynthesize new cytochromes c

Cytochrome c (cyt c), required for electron transport in mitochondria, possesses a covalently attached heme cofactor. Attachment is catalyzed by holocytochrome c synthase (HCCS), leading to two thioether bonds between heme and a conserved CXXCH motif of cyt c In cyt c, histidine (His19) of CXXCH acts as an axial ligand to heme iron and upon release of holocytochrome c from HCCS, folding leads to formation of a second axial interaction with methionine (Met81). We previously discovered mutations in human HCCS that facilitate increased biosynthesis of cyt c in recombinant Escherichia coli Focusing on HCCS E159A, novel cyt c variants in quantities that are sufficient for biophysical analysis are biosynthesized. Cyt c H19M, the first bis-Met liganded cyt c, is compared with other axial ligand variants (M81A, M81H) and single thioether cyt c variants. For variants with axial ligand substitutions, electronic absorption, near-UV circular dichroism, and electron paramagnetic resonance spectroscopy provide evidence that axial ligands are changed and the heme environment is altered. Circular dichroism spectra in far UV and thermal denaturation analyses demonstrate that axial ligand changes do not affect secondary structures and stability. Redox potentials span a 400-mV range (+349 mV vs. standard hydrogen electrode, H19M; +252 mV, WT; -19 mV, M81A; -69 mV, M81H). We discuss the results in the context of a four-step mechanism for HCCS, whereby HCCS mutants such as E159A are enhanced in release (step 4) of cyt c from the HCCS active site; thus, we term these "release mutants."

Rigidifying flexible sites: An approach to improve stability of chondroitinase ABC I

The stability of chondroitin ABC lyase I (cABC I) at physiological temperature is one of the current obstacles to its clinical application. In this study, we used a protein engineering approach; rigidify flexible sites, to improve stability of cABC I. B-factor analysis showed a flexible loop at the N-terminal domain of cABC I which may be involved in its thermal instability and five residues in this region were replaced with proline. Thermal inactivation and thermal denaturation analysis revealed that Glu138Pro mutation increased half-life and Tm of enzyme, respectively. The Km values of mutated enzymes were slightly increased compared to the wild type enzyme. The results of limited proteolysis indicated that Glu138Pro mutant was more resistant against trypsinolysis and this variant was less quenched in both acrylamide and KI quenching experiments. Moreover, intrinsic fluorescence intensity of Glu138Pro variant was increased and its ANS fluorescence intensity was decreased, whereas no considerable changes were observed in the far-UV CD spectra. The structural analyses indicated compactness of structure of Glu138Pro enzyme which can be related to moderately enhanced stability of this mutant. This study demonstrated that rigidifying flexible residues can be considered as a possible approach to increase the stability of the protein.