Thermal Denaturation Experiments Research Articles

An intronic RNA structure modulates expression of the mRNA biogenesis factor Sus1.

Sus1 is a conserved protein involved in chromatin remodeling and mRNA biogenesis. Unlike most yeast genes, the SUS1 pre-mRNA of Saccharomyces cerevisiae contains two introns and is alternatively spliced, retaining one or both introns in response to changes in environmental conditions. SUS1 splicing may allow the cell to control Sus1 expression, but the mechanisms that regulate this process remain unknown. Using in silico analyses together with NMR spectroscopy, gel electrophoresis, and UV thermal denaturation experiments, we show that the downstream intron (I2) of SUS1 forms a weakly stable, 37-nucleotide stem–loop structure containing the branch site near its apical loop and the 3′ splice site after the stem terminus. A cellular assay revealed that two of four mutants containing altered I2 structures had significantly impaired SUS1 expression. Semiquantitative RT-PCR experiments indicated that all mutants accumulated unspliced SUS1 pre-mRNA and/or induced distorted levels of fully spliced mRNA relative to wild type. Concomitantly, Sus1 cellular functions in histone H2B deubiquitination and mRNA export were affected in I2 hairpin mutants that inhibited splicing. This work demonstrates that I2 structure is relevant for SUS1 expression, and that this effect is likely exerted through modulation of splicing.

PrP charge structure encodes interdomain interactions.

Almost all proteins contain charged residues, and their chain distribution is tailored to fulfill essential ionic interactions for folding, binding and catalysis. Among proteins, the hinged two-domain chain of the cellular prion protein (PrPC) exhibits a peculiar charge structure with unclear consequences in its structural malleability. To decipher the charge design role, we generated charge-reverted mutants for each domain and analyzed their effect on conformational and metabolic features. We found that charges contain the information for interdomain interactions. Use of dynamic light scattering and thermal denaturation experiments delineates the compaction of the α-fold by an electrostatic compensation between the polybasic 23–30 region and the α3 electronegative surface. This interaction increases stability and disfavors fibrillation. Independently of this structural effect, the N-terminal electropositive clusters regulate the α-cleavage efficiency. In the fibrillar state, use of circular dichroism, atomic-force and fluorescence microscopies reveal that the N-terminal positive clusters and the α3 electronegative surface dictate the secondary structure, the assembly hierarchy and the growth length of the fibril state. These findings show that the PrP charge structure functions as a code set up to ensure function and reduce pathogenic routes.

Effect of helix length on the stability of the Lac repressor antiparallel coiled coil.

The helix length dependence of the stability of antiparallel four-chain coiled coils is investigated using eight synthetic peptides (Lac21-Lac28) whose sequences are derived from the tetramerization domain of the Lac repressor protein. Previous studies using analytical ultracentrifugation sedimentation equilibrium experiments to characterize Lac21 and Lac28 justifies the use of a two state model to describe the unfolding behavior of these two peptides. Using circular dichroism spectropolarimetry as a measure of tetramer assembly, both chemical and thermal denaturation experiments were carried out to determine thermodynamic parameters. We found that the hydrophobic core residues provide the greatest impact on stability and, as a consequence, must reorganize the register of the antiparallel helices to accommodate the burial of the nonpolar amino acids. Addition of noncore residues appears to have only a minor effect on stability, and in some cases, show a slight destabilization.

Recognition of Double-Stranded DNA Using Energetically Activated Duplexes Modified with N2'-Pyrene-, Perylene-, or Coronene-Functionalized 2'-N-Methyl-2'-amino-DNA Monomers.

Invader probes have been proposed as alternatives to polyamides, triplex-forming oligonucleotides, and peptide nucleic acids for recognition of chromosomal DNA targets. These double-stranded probes are activated for DNA recognition by +1 interstrand zippers of pyrene-functionalized nucleotides. This particular motif forces the intercalating pyrene moieties into the same region, resulting in perturbation and destabilization of the probe duplex. In contrast, the two probe strands display very high affinity toward complementary DNA. The energy difference between the probe duplexes and recognition complexes provides the driving force for DNA recognition. In the present study, we explore the properties of Invader probes based on larger intercalators, i.e., perylene and coronene, expecting that the larger π-surface area will result in additional destabilization of the probe duplex and further stabilization of probe-target duplexes, in effect increasing the thermodynamic driving force for DNA recognition. Toward this end, we developed protocols for 2'-N-methyl-2'-amino-2'-deoxyuridine phosphoramidites that are functionalized at the N2'-position with pyrene, perylene, or coronene moieties and incorporated these monomers into oligodeoxyribonucleotides (ONs). The resulting ONs and Invader probes are characterized by thermal denaturation experiments, analysis of thermodynamic parameters, absorption and fluorescence spectroscopy, and DNA recognition experiments. Invader probes based on large intercalators efficiently recognize model targets.

Dicationic derivatives of dinaphthotetraaza[14]annulene: synthesis, crystal structures and the preliminary evaluation of their DNA binding properties

Four new water-soluble, fluorescent derivatives of dinaphthotetraaza[14]annulene (DNTAA) have been synthesized varying in the structure, dimensions and spatial arrangements of their meso side groups. The products have been carefully characterized by elemental analyses, spectroscopy, crystal structures and quantum-chemical calculations employing DFT methodology. One representative product of the DNTAA series was tested for DNA binding using UV–vis titrations, CD and thermal denaturation experiments. Intercalation of ds-DNA was recognized as a dominant binding mode. In contrast to non-fluorescent phenylene analogues (DBTAA), reported previously, DNTAA derivative showed threefold fluorescence increase upon DNA binding, and offered intriguing new applications as a fluorescent DNA/RNA dye.

Sensing of Double-Stranded DNA/RNA Secondary Structures by Water Soluble Homochiral Perylene Bisimide Dyes.

A broad series of homochiral perylene bisimide (PBI) dyes were synthesized that are appended with amino acids and cationic side chains at the imide positions. Self-assembly behavior of these ionic PBIs has been studied in aqueous media by UV/Vis spectroscopy, revealing formation of excitonically coupled H-type aggregates. The interactions of these ionic PBIs with different ds-DNA and ds-RNA have been explored by thermal denaturation, fluorimetric titration and circular dichroism (CD) experiments. These PBIs strongly stabilized ds-DNA/RNA against thermal denaturation as revealed by high melting temperatures of the formed PBI/polynucleotide complexes. Fluorimetric titrations showed that these PBIs bind to ds-DNA/RNA with high binding constants depending on the number of the positive charges in the side chains. Thus, spermine-containing PBIs with six positive charges each showed higher binding constants (logKs =9.2-9.8) than their dioxa analogues (logKs =6.5-7.9) having two positive charges each. Induced circular dichroism (ICD) of PBI assemblies created within DNA/RNA grooves was observed. These ICD profiles are strongly dependent on the steric demand of the chiral substituents of the amino acid units and the secondary structure of the DNA or RNA. The observed ICD effects can be explained by non-covalent binding of excitonically coupled PBI dimer aggregates into the minor groove of DNA and major groove of RNA which is further supported by molecular modeling studies.

Thermodynamic Examination of the U6 ISL RNA Bulge

Spliceosome is large complex made up of five snRNA and nearly 70 proteins that assembles on pre‐mRNA to excise the introns. The U6 subunit of the spliceosome is required for spliceosome assembly and activation. Spliceosome undergoes number of structural rearrangements that disrupt U4/U6 complex and form U2/U6 complex to become activated. The association of U2/U6 complex results in the formation of a highly conserved internal stem loop (ISL) in U6 RNA. The formation of a 1x2 nucleotide bulge that binds catalytically active Mg2+ at U80 position is essential in the RNA cleavage. The 1x2 bulge contains an A79•C67 non‐canonical base pair, which forms a stronger A+•C base pair in lower pH conditions. Upon the A+•C base pair formation, the ISL adopts a conformation closer to an A‐form helix and the unbound U80 in the bulge is forced out into the major groove. This conformation is mutually antagonistic to the binding of the catalytic Mg2+ ion, and therefore prevents splicing activity. Thermal denaturation experiments were performed on RNA constructs derived to mimic 1x2 bulge of the U6 RNA. We tested constructs containing the conserved bulged nucleotides as well as those with modifications that disrupt spliceosomal function. All constructs were examined in 1 M KCl and in varying Mg2+ concentrations at pH of 5.5 and 7.5. The thermodynamic contributions of the bulged RNA were compared to those of the helical RNA and DNA constructs. Thermodynamics of RNA with modification in and around the 1x2 bulge loop will be presented and influence of pH and ionic conditions will be discussed.

Analytical Characterization of FGF Signaling Complex

Fibroblast growth factors (FGF) are a family of growth hormones that act as regulators in key biological processes such as angiogenesis, wound healing, embryonic development and select endocrine signaling pathways. It is currently theorized that the mechanism of the interaction of FGF to their receptors, fibroblast growth factor receptors (FGFR) is greatly reliant upon the binding of heparin to FGFs. It is theorized that the D2 domain is the primary site for the FGF bind and interaction. The objective of this study is to characterize the structure of the FGF-D2 domain. A structural analog of heparin, sucrose octasulfate (SOS), is employed in this study to determine the role of heparin in the formation of the FGF-FGFR complex. Isothermal titration calorimetry experiments were preformed to assess the binding affinity of FGF and the D2 domain, these experiments indicate that human acidic FGF (FGF-1) binds to the D2 domain with high affinity in both the presence and absence of SOS (Kd(appa) ∼ 10−7 M and Kd(appa) ∼ 10−8 M respectively). Thermal denaturation experiments, monitored by far-UV CD, indicate that both FGF-1 and D2 domain undergo slight conformational changes as a result of binding and elucidates the presence of SOS stabilizes a 1:1 FGF-D2 domain complex. The FGF signaling complex, comprised of the FGF-1, the D2 domain, and SOS, structure elucidated by X-ray crystallography indicated that the complex exists as a 2:2:2 symmetrical conjugation. Analysis via 2D 1H and 15N NMR chemical shift perturbation (CSP) has allowed for the accurate mapping of the SOS and FGF binding sites of the D2 domain. The data resulting from this study suggest that the primary role of heparin in the FGF signaling process is limited to stabilizing the FGF-FGFR complex.

DNA binding, photo-induced DNA cleavage and cytotoxicity studies of lomefloxacin and its transition metal complexes

This work was focused on a study of the DNA binding and cleavage properties of lomefloxacin (LMF) and its ternary transition metal complexes with glycine. The nature of the binding interactions between compounds and calf thymus DNA (CT-DNA) was studied by electronic absorption spectra, fluorescence spectra and thermal denaturation experiments. The obtained results revealed that LMF and its complexes could interact with CT-DNA via partial/moderate intercalative mode. Furthermore, the DNA cleavage activities of the compounds were investigated by gel electrophoresis. Mechanistic studies of DNA cleavage suggest that singlet oxygen (1O2) is likely to be the cleaving agent via an oxidative pathway, except for Cu(II) complex which proceeds via both oxidative and hydrolytic pathways. Antimicrobial and antitumor activities of the compounds were also studied against some kinds of bacteria, fungi and human cell lines.

Correlation of heme binding affinity and enzyme kinetics of dehaloperoxidase.

Chemical and thermal denaturation of dehaloperoxidase-hemoglobin (DHP) was investigated to test the relative stability of isoforms DHP A and DHP B and the H55V mutant of DHP A with respect to heme loss. In thermal denaturation experiments, heme loss was observed at temperatures of 54, 46, and 61 °C in DHP A, DHP B, and H55V, respectively. Guanidinium hydrochloride (GdnHCl)- and urea-induced denaturation was observed at respective concentrations of 1.15 ± 0.01 M DHP A and 1.09 ± 0.02 M DHP B, and 5.19 ± 0.05 M DHP A and 4.12 ± 0.14 M DHP B, respectively. The binding affinity of heme appears to be significantly smaller in both isoforms of DHP than in myoglobins. This observation was corroborated by heme transfer experiments, in which heme was observed to transfer for DHP A and B to horse skeletal muscle myoglobin (HSMb). GdnHCl-induced denaturation suggests a threshold of 1 mM for stabilization by binding of the inhibitor 4-bromophenol (4-BP). Concentrations of 4-BP greater than 1 mM caused destabilization. Urea-induced denaturation showed only destabilizing effects from phenolic ligand binding. Heme transfer experiments from DHP to HSMb further support the hypothesis that the binding of halophenols to DHP facilitates the removal of the heme. Thermal denaturation assessed via UV-visible spectroscopy and that assessed by differential scanning calorimetry (DSC) are both in agreement with chemical denaturation experiments and show that the denaturing abilities of the halophenols improve with the size of the para halogen atom in 4-XP, where X = iodo, bromo, chloro, or fluoro (4-IP > 4-BP > 4-CP > 4-FP), and the number of halo substituents as in 2,4,6-tribromophenol (2,4,6-TBP > 4-BP). DHP B, which differs in five amino acids, is less stable than DHP A with ΔHcal and Tm values of 165.1 kJ/mol and 47.5 °C compared to values of 183.3 kJ/mol and 50.4 °C for DHP B and DHP A, respectively. Kinetic studies verified that DHP B has a catalytic efficiency (kcat/Km) ∼5-6 times greater than that of DHP A but showed an increased level of substrate inhibition in DHP B for both 2,4,6-TCP and 2,4,6-TBP. An inverse correlation between protein stability with respect to heme loss and catalytic efficiency is suggested on the basis of the fact that the heme in DHP B has a stability lower than that of DHP A but a catalytic efficiency higher than that of DHP A.

Identification of a surface glutamine residue (Q64) of Escherichia coli EntA required for interaction with EntE

The enterobactin biosynthetic enzyme EntA forms a complex with EntE, the next enzyme in the pathway, to enhance activation of the enterobactin precursor 2,3-dihydroxybenzoate. Here we used phage display to identify an EntE-interacting region on the surface of EntA. Upon panning immobilized EntE with a random peptide phage library, we recovered 47 unique EntE-binding dodecamer peptide sequences that aligned to a region of the EntA primary sequence corresponding to helix α4. In order to further investigate this region, we mutagenized EntA Q64, a hydrogen-bonding residue found on the surface-exposed face α4. Far-UV circular dichroism, thermal denaturation experiments, and enzymatic assays showed that mutation of EntA residue Gln 64 to alanine (Q64A) had no deleterious effect on EntA structure or function. By following near-UV CD spectral changes, we found that the spectrum of wild-type EntA was altered in the presence of EntE, indicative of conformational changes in EntA aromatic chromophores upon formation of the EntA–EntE complex. However, EntE did not affect the CD spectrum of EntA variant Q64A, demonstrating that this variant did not interact with EntE in a manner similar to wild-type EntA. Analytical ultracentrifugation of wild-type and variant EntA proteins showed that EntA Q64A was predominantly dimeric at 20μM, unlike wild-type EntA which was predominantly tetrameric. Taken together, our findings establish that EntA α4 is required for efficient formation of the EntA–EntE as well as for EntA oligomerization.

Unexpected DNA affinity and sequence selectivity through core rigidity in guanidinium-based minor groove binders.

In this paper we report the design and biophysical evaluation of novel rigid-core symmetric and asymmetric dicationic DNA binders containing 9H-fluorene and 9,10-dihydroanthracene cores as well as the synthesis of one of these fluorene derivatives. First, the affinity toward particular DNA sequences of these compounds and flexible core derivatives was evaluated by means of surface plasmon resonance and thermal denaturation experiments finding that the position of the cations significantly influence the binding strength. Then their affinity and mode of binding were further studied by performing circular dichroism and UV studies and the results obtained were rationalized by means of DFT calculations. We found that the fluorene derivatives prepared have the ability to bind to the minor groove of certain DNA sequences and intercalate to others, whereas the dihydroanthracene compounds bind via intercalation to all the DNA sequences studied here.

Gemini surfactants affect the structure, stability, and activity of ribonuclease Sa.

Gemini surfactants have important advantages, e.g., low micromolar CMCs and slow millisecond monomer ↔ micelle kinetics, for membrane mimetics and for delivering nucleic acids for gene therapy or RNA silencing. However, as a prerequisite, it is important to characterize interactions occurring between Gemini surfactants and proteins. Here NMR and CD spectroscopies are employed to investigate the interactions of cationic Gemini surfactants with RNase Sa, a negatively charged ribonuclease. We find that RNase Sa binds Gemini surfactant monomers and micelles at pH values above 4 to form aggregates. Below pH 4, where the protein is positively charged, these aggregates dissolve and interactions are undetectable. Thermal denaturation experiments show that surfactant lowers RNase Sa's conformational stability, suggesting that surfactant binds the protein's denatured state preferentially. Finally, Gemini surfactants were found to bind RNA, leading to the formation of large complexes. Interestingly, Gemini surfactant binding did not prevent RNase Sa from cleaving RNA.

Synthesis, Characterization and DNA Interaction Studies of (N,N'-Bis(5-phenylazosalicylaldehyde)-ethylenediamine) Cobalt(II) Complex

In the present study, at first, azo Schiff base ligand of (N,N'-bis(5-phenylazosalicylaldehyde)-ethylenediamine) (H2L) has been synthesized by condensation reaction of 5-phenylazosalicylaldehyde and ethylenediamine in 2:1 molar ratio, respectively. Then, its cobalt complex (CoL) was synthesized by reaction of Co(OAc)2·4H2O with ligand (H2L) in 1:1 molar ratio in ethanol solvent. This ligand and its cobalt complex containing azo functional groups were characterized using elemental analysis, 1 H-NMR, UV-vis and IR spectroscopies. Subsequently, the interaction between native calf thymus deoxyribonucleic acid (ct-DNA) and CoL complex was investigated in 10 mM Tris/HCl buffer solution, pH = 7 using UV-vis absorption, thermal denaturation technique and viscosity measurements. From spectrophotometric titration experiments, the binding constant of CoL complex with ct-DNA was found to be (2.4 ± 0.2) × 10 4 M �1 . The thermodynamic parameters were calculated by van’t Hoff equation.The enthalpy and entropy changes were 5753.94 ± 172.66 kcal/mol and 43.93 ± 1.18 cal/mol·K at 25 °C, respectively. Thermal denaturation experiments represent the increasing of melting temperature of ct-DNA (about 0.93 °C) due to binding of CoL complex. The results indicate that the process is entropy- driven and suggest that hydrophobic interactions are the main driving force for the complex formation.

Structure-based classification of ice-binding proteins

Since the antifreeze protein (AFP) super family has low structural identity, classification standard of the AFPs is presently ambiguous. Newly identified ice-binding proteins (IBPs), named so after the function of the AFPs, have similar structural identity and function that interact to the ice. Identification and characterization of IBPs from the eukaryotic microorganisms Typhulaishikariensis (TisAFP) and Leucosporidium sp. (LeIBP) revealed that both are glycosylated and have irregular motif on the ice-binding site (IBS). The IBPs share a unique right-handed β-helix, which provides an advantage of broad-range interaction surface. The other IBP encoded by the Antarctic bacterium Flavobacterium frigoris PSI was determined at 2.1-Å resolution to gain insight into its ice-binding mechanism. The structure of FfIBP shows the presence of an intra-molecular disulfide bond in the loop region between α2 and α4 (capping region), unlike that of LeIBP and TisAFP. Electron density for this disulfide bond was seen between Cys107 and Cys124 during the structure refinement process and the Cβ–Cβ distance between Cys107 and Cys124 was 3.9 Å. By sequence alignments and structural comparisons of IBPs, we defined two groups within IBPs, depending on the sequence differences between the α2 and α4 loop regions and the presence of the disulfide bond. In addition, to investigate the effects of the capping region on the activity and stability of FfIBP, we determined the crystal structure and measured the thermal stability of mutants that swapped the capping region of FfIBP and LeIBP (mFfIBP and mLeIBP). In thermal denaturation experiments, it is clear that the capping-head region of FfIBP is more stable than that of LeIBP and is important for the overall stability of IBP, although it is not directly involved in the antifreeze activity.

Understanding the guanidine-like cationic moiety for optimal binding into the DNA minor groove.

Based on our previous positive results with bis-guanidine-like diaromatic compounds as DNA minor groove binders, we propose a new family: bis-2-amino-1,4,5,6-tetrahydropyrimidines. According to calculated parameters, these dicationic systems would have a more suitable size and lipophilicity for binding into the minor groove than previous series. Moreover, their DFT-optimised structures and docking into an AT oligomer model show that they would bind in the minor groove with good strength and without energy penalty. Hence, we prepared compounds 4 a-c and evaluated their binding to ssDNA and poly(dA-dT)2 by thermal denaturation experiments. The results showed that 4 a (CO) and 4 d (NH) were the best DNA binders. Compared to the previous series, 4 a-d are better binders than bis-guanidiniums but poorer than bis-2-aminoimidazolinium derivatives. Moreover, circular dichroism experiments using ssDNA and poly(dA-dT)2 confirmed binding into the minor groove. Based on our computational design as well as biophysical studies, we have been able to determine that the optimal interaction of guanidine-like dications in the minor grove occurs with bis-2-aminoimidazolinium systems.

Characterization of reconstituted high-density lipoprotein particles formed by lipid interactions with human serum amyloid A

The acute-phase human protein serum amyloid A (SAA) is enriched in high-density lipoprotein (HDL) in patients with inflammatory diseases. Compared with normal HDL containing apolipoprotein A-I, which is the principal protein component, characteristics of acute-phase HDL containing SAA remain largely undefined. In the present study, we examined the physicochemical properties of reconstituted HDL (rHDL) particles formed by lipid interactions with SAA. Fluorescence and circular dichroism measurements revealed that although SAA was unstructured at physiological temperature, α-helix formation was induced upon binding to phospholipid vesicles. SAA also formed rHDL particles by solubilizing phospholipid vesicles through mechanisms that are common to other exchangeable apolipoproteins. Dynamic light scattering and nondenaturing gradient gel electrophoresis analyses of rHDL after gel filtration revealed particle sizes of approximately 10nm, and a discoidal shape was verified by transmission electron microscopy. Thermal denaturation experiments indicated that SAA molecules in rHDL retained α-helical conformations at 37°C, but were almost completely denatured around 60°C. Furthermore, trypsin digestion experiments showed that lipid binding rendered SAA molecules resistant to protein degradation. In humans, three major SAA1 isoforms (SAA1.1, 1.3, and 1.5) are known. Although these isoforms have different amino acids at residues 52 and 57, no major differences in physicochemical properties between rHDL particles resulting from lipid interactions with SAA isoforms have been found. The present data provide useful insights into the effects of SAA enrichment on the physicochemical properties of HDL.

Selective stabilization of abasic site-containing DNA by insertion of sterically demanding biaryl ligands.

Biaryl derivatives that consist of one DNA-intercalating unit and a sterically demanding component exhibit a specific behavior towards abasic site-containing DNA (AP-DNA) as determined by thermal DNA denaturation experiments, spectrometric titrations and CD spectroscopic analysis. Specifically, these ligands strongly stabilize AP-DNA towards dissociation, whereas they do not or only marginally affect the melting temperature of regular duplex DNA.

Efficient synthesis of an indoloquinolizinium alkaloid selective DNA-binder by ring-closing metathesis.

Two total syntheses of the indolo[2,3-a]quinolizinium cation have been accomplished through the application of two ring-closing metathesis reactions to form the pyridinium ring. One of these approaches provides the tetracyclic cation in only five steps from commercially available harmane. Fluorescence-based thermal denaturation experiments, as well as spectrofluorimetric titration, circular dichroism measurements, and theoretical simulations, showed a consistent DNA-binding capacity by intercalation with a marked preference for AT-rich sequences.

NMR structure of an ethylene interstrand cross-linked DNA which mimics the lesion formed by 1,3-bis(2-chloroethyl)-1-nitrosourea.

The bisalkylating agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), used in cancer chemotherapy to hinder cellular proliferation, forms lethal interstrand cross-links (ICLs) in DNA. BCNU generates an ethylene linkage connecting the two DNA strands at the N1 atom of 2'-deoxyguanosine and N3 atom of 2'-deoxycytidine, which is a synthetically challenging probe to prepare. To this end, an ICL duplex linking the N1 atom of 2'-deoxyinosine to the N3 atom of thymidine via an ethylene linker was devised as a mimic. We have solved the structure of this ICL duplex by a combination of molecular dynamics and high-field NMR experiments. The ethylene linker is well-accommodated in the duplex with minimal global and local perturbations relative to the unmodified duplex. These results may account for the substantial stabilization of the ICL duplex observed by UV thermal denaturation experiments and provides structural insights of a probe that may be useful for DNA repair studies.