Studies on the formation and stability of triplex DNA using fluorescence correlation spectroscopy

Triplex DNA has become one of the most useful recognition motifs in the design of new molecular biology tools, therapeutic agents and sophisticated DNA‐based nanomaterials because of its direct recognition of natural double‐stranded DNA. In this paper, we developed a sensitive and microscale method to study the formation and stability characterization of triplex DNA using fluorescence correlation spectroscopy (FCS). The principle of this method is mainly based on the excellent capacity of FCS for sensitively distinguishing between free single‐strand DNA (ssDNA) fluorescent probes and fluorescent probe–double‐strand DNA (dsDNA) hybridized complexes. First, we systematically investigated the experimental conditions of triplex DNA formation. Then, we evaluated the equilibrium association constants (K_a) under different ssDNA probe lengths, composition and pH. Finally, we used FCS to measure the hybridization fraction of a 20‐mer perfectly matched ssDNA probe and three single‐base mismatched ssDNA probes with 146‐mer dsDNA. Our data illustrated that FCS is a useful tool for the direct determination of the thermodynamic parameters of triplex DNA formation and discrimination of a single‐base mismatch of triplex DNA without denaturation. Compared with current methods, our method is characterized by high sensitivity, good universality and small sample and reagent requirements. More importantly, our method has the potential to become a platform for triplex DNA research in vitro. Copyright © 2015 John Wiley & Sons, Ltd.     

The structural analysis and the role of calcium binding site for thermal stability in mannanase

Mannanase is an important enzyme involved in the degradation of mannan, production of bioactive oligosaccharides, and biobleaching of kraft pulp. Mannanase must be thermostable for use in industrial applications. In a previous study, we found that the thermal stability of mannanase from Streptomyces thermolilacinus (StMan) and Thermobifida fusca (TfMan) is enhanced by calcium. Here, we investigated the relationship between the three-dimensional structure and primary sequence to identify the putative calcium-binding site. The results of site-directed mutagenesis experiments indicated that Asp-285, Glu-286, and Asp-287 of StMan (StDEDAAAdC) and Asp-264, Glu-265, and Asp-266 of TfMan (TfDEDAAAdC) were the key residues for calcium binding affinity. Isothermal titration calorimetry revealed that the catalytic domain of StMan and TfMan (StMandC and TfMandC, respectively) bound calcium with a K_a of 3.02 × 10⁴ M⁻¹ and 1.52 × 10⁴ M⁻¹, respectively, both with stoichiometry consistent with one calcium-binding site per molecule of enzyme. Non-calcium-binding mutants (StDEDAAAdC and TfDEDAAAdC) did not show any calorimetric change. From the primary structure alignment of several mannanases, the calcium-binding site was found to be highly conserved in GH5 bacterial mannanases. This is the first study indicating enhanced thermal stability of GH5 bacterial mannanases by calcium binding.     

A comparison of the different DNA binding specificities of the bZip proteins C/EBP and GCN4.

The bZip proteins GCN4 and C/EBP differ in their DNA binding specificities: GCN4 binds well to the pseudopalindromic AP1 site 5'-A4T3G2A1C0T1C2'A3'T4'-3' and to the palindromic ATF/CREB sequence 5'-A4T3G2A1-C0*G0'T1'C2'A3'T4'-3'; C/EBP preferentially recognizes the palindromic sequence 5'-A4T3T2G1C0*G0'C1'A2'-A3'T4'-3'. According to the X-ray structures of GCN4-DNA complexes, five residues of the basic region of GCN4 are involved in specific base contacts: asparagine -18, alanine -15, alanine -14, serine -11 and arginine -10 (numbered relative to the start point of the leucine zipper, which we define as +1). In the basic region of C/EBP position -14 is occupied by valine instead of alanine, the other four residues being identical. Here we analyse the role of valine -14 in C/EBP-DNA complex formation. Starting from a C/EBP-GCN4 chimeric bZip peptide which displays C/EBP specificity, we systematically mutated position -14 of its basic region and characterized the DNA binding specificities of the 20 possible different peptides by gel mobility shift assays with various target sites. We present evidence that valine -14 of C/EBP interacts more strongly with thymine 2 than with cytosine 1' of the C/EBP binding site, unlike the corresponding alanine -14 of GCN4, which exclusively contacts thymine 1' of the GCN4 binding sites.     

A novel DNA extraction and duplex polymerase chain reaction assay for the rapid detection of Prototheca zopfii genotype 2 in milk

Aims: To develop a PCR‐based assay to detect Prototheca zopfii (P. zopfii) and its mastitis‐related subtype (genotype 2) directly from milk samples. Methods and Results: The DNA extraction method herein is based on the lysing properties of chemical agents, mechanical grinding and DNA‐binding properties of silica particles; this method was developed to rapidly extract DNA directly from P. zopfii in bovine milk. Two pairs of primers specific for P. zopfii and genotype 2 were used in the duplex PCR, and a sensitivity test showed that the detection level was 5 × 10² colony‐forming units (CFU) ml^?1 for P. zopfii and 5 × 10³ CFU ml^?1 for genotype 2. Furthermore, a practical survey of 23 milk samples showed that the assay produced results that were in accordance with those obtained by the conventional microbiology method. Conclusions: The DNA extraction method is effective in isolating sufficient quantities of DNA from P. zopfii in milk for PCR analysis. The PCR assay is economical, sensitive and more rapid than the conventional culture method. Significance and Impact of the Study: The assay could be used as an alternative method for the rapid the detection of bovine mastitis resulting from P. zopfii genotype 2.     

DNA binding mode transitions of Escherichia coli HU(alphabeta): evidence for formation of a bent DNA--protein complex on intact, linear duplex DNA

Escherichia coli HU_αβ, a major nucleoid-associated protein, organizes chromosomal DNA and facilitates numerous DNA transactions. Using isothermal titration calorimetry, fluorescence resonance energy transfer and a series of DNA lengths (8 bp, 15 bp, 34 bp, 38 bp and 160 bp) we established that HU_αβ interacts with duplex DNA using three different nonspecific binding modes. Both the HU to DNA molar ratio ([HU]/[DNA]) and DNA length dictate the dominant HU binding mode. On sufficiently long DNA (≥ 34 bp), at low [HU]/[DNA], HU populates a noncooperative 34 bp binding mode with a binding constant of 2.1 ± 0.4 × 10⁶ M^− 1, and a binding enthalpy of + 7.7 ± 0.6 kcal/mol at 15 °C and 0.15 M Na⁺. With increasing [HU]/[DNA], HU bound in the noncooperative 34 bp mode progressively converts to two cooperative (ω∼20) modes with site sizes of 10 bp and 6 bp. These latter modes exhibit smaller binding constants (1.1 ± 0.2 × 10⁵ M^− 1 for the 10 bp mode, 3.5 ± 1.4 × 10⁴ M^− 1 for the 6 bp mode) and binding enthalpies (4.2 ± 0.3 kcal/mol for the 10 bp mode, − 1.6 ± 0.3 kcal/mol for the 6 bp mode). As DNA length increases to 34 bp or more at low [HU]/[DNA], the small modes are replaced by the 34 bp binding mode. Fluorescence resonance energy transfer data demonstrate that the 34 bp mode bends DNA by 143 ± 6° whereas the 6 bp and 10 bp modes do not. The model proposed in this study provides a novel quantitative and comprehensive framework for reconciling previous structural and solution studies of HU, including single molecule (force extension measurement), fluorescence, and electrophoretic gel mobility-shift assays. In particular, it explains how HU condenses or extends DNA depending on the relative concentrations of HU and DNA.     

Brefeldin A inhibits expression of DNA packaging proteins and nucleocapsid formation of human cytomegalovirus

In this study we used the fungal antibiotic brefeldin A (BFA) to analyze its effect on viral replication. Analysis by electron microscopy demonstrated that no viral particles were observed in cells treated before the onset of viral replication. In the presence of BFA expression of IE2, MCP, pUL104, pUL56 and pUL89 were reduced, while no or slight effect was observed on expression of pp65, pUL44 and pUL57. Strikingly, real time PCR revealed that de novo viral DNA synthesis is reduced but not completely abolished in the presence of BFA. These results indicated that BFA represents a multi-functional compound leading to inhibition of several steps of viral maturation such as expression of viral DNA packaging proteins and capsid formation.     

Both helical propensity and side-chain hydrophobicity at a partially exposed site in alpha-helix contribute to the thermodynamic stability of ubiquitin

Pfam family DUF1023 consists entirely of uncharacterized proteins generated by sequencing the genomes of Actinobacteria (Bateman A., et al., Nucleic Acids Res. 2004;32 Database issue:D138-141.) Utilizing sequence similarity detection methods, we infer homology between DUF1023 and alpha/beta hydrolases. DUF1023 proteins conserve the core secondary structures in alpha/beta hydrolase fold, and share similar catalytic machinery as that of alpha/beta hydrolases. We predict DUF1023 spatial structure and deduce that they function as hydrolases utilizing catalytic Ser-His-Asp triad with the serine as a nucleophile.     

Thermodynamic signature of GCN4-bZIP binding to DNA indicates the role of water in discriminating between the AP-1 and ATF/CREB sites

The energetic basis of GCN4-bZIP complexes with the AP-1 and ATF/CREB sites was investigated by optical methods and scanning and isothermal titration microcalorimetry. The dissociation constant of the bZIP dimer was found to be significantly higher than that of its isolated leucine zipper domain: at 20 degrees C it is 1.45microM and increases with temperature. To avoid complications from dissociation of this dimer, DNA binding experiments were carried out using an SS crosslinked version of the bZIP. The thermodynamic characteristics of the bZIP/DNA association measured at different temperatures and salt concentrations were corrected for the contribution of refolding the basic segment upon binding, determined from the scanning calorimetric experiments. Fluorescence anisotropy titration experiments showed that the association constants of the bZIP at 20 degrees C with the AP-1 and ATF/CREB binding sites do not differ much, being 1.5nM and 6.4nM, corresponding to Gibbs energies of -49kJmol(-1) and -46kJmol(-1), respectively. Almost half of the Gibbs energy is attributable to the electrostatic component, resulting from the entropic effect of counterion release upon DNA association with the bZIP and is identical for both sites. In contrast to the Gibbs energies, the enthalpies of association of the fully folded bZIP with the AP-1 and ATF/CREB sites, and correspondingly the entropies of association, are very different. bZIP binding to the AP-1 site is characterized by a substantially larger negative enthalpy and non-electrostatic entropy than to the ATF/CREB site, implying that the AP-1 complex incorporates significantly more water molecules than the ATF/CREB complex.     

Impact of bulge loop size on DNA triplet repeat domains: Implications for DNA repair and expansion

Repetitive DNA sequences exhibit complex structural and energy landscapes, populated by metastable, noncanonical states, that favor expansion and deletion events correlated with disease phenotypes. To probe the origins of such genotype–phenotype linkages, we report the impact of sequence and repeat number on properties of (CNG) repeat bulge loops. We find the stability of duplexes with a repeat bulge loop is controlled by two opposing effects; a loop junction‐dependent destabilization of the underlying double helix, and a self‐structure dependent stabilization of the repeat bulge loop. For small bulge loops, destabilization of the underlying double helix overwhelms any favorable contribution from loop self‐structure. As bulge loop size increases, the stabilizing loop structure contribution dominates. The role of sequence on repeat loop stability can be understood in terms of its impact on the opposing influences of junction formation and loop structure. The nature of the bulge loop affects the thermodynamics of these two contributions differently, resulting in unique differences in repeat size‐dependent minima in the overall enthalpy, entropy, and free energy changes. Our results define factors that control repeat bulge loop formation; knowledge required to understand how this helix imperfection is linked to DNA expansion, deletion, and disease phenotypes. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 1–12, 2014.     

The effect of mutations on peptide models of the DNA binding helix of p53: Evidence for a correlation between structure and tumorigenesis

The tumor suppresser protein p53 has been called the “guardian of the genome.” DNA damage induces p53 to either halt the cell cycle, allowing for repair, or initiate apoptosis. P53 is mutated in over 50% of human tumors and it has been proposed that many tumorigenic mutations are deleterious to p53 because they induce local unfolding. To explore this hypothesis, peptide models have been developed to study tumorigenic mutations in the H2 helix of the p53 core domain. This helix is rich with charged residues and is a key component of the DNA binding region. A 16‐residue peptide corresponding to the H2 wild‐type sequence extended with an Ala‐rich C‐terminus was synthesized and studied by ¹H‐nmr (500 MHz) and CD. The nmr studies demonstrate that this peptide adopts helical structure in solution. Six additional peptides corresponding to subtle tumorigenic mutations were synthesized and CD was used to assess the relative stability of these “mutant analogues.” All six mutations studied are destabilizing relative to the wild type, with ΔΔG values in the range of 0.26 to 1.35 kcal mol⁻¹. Surprisingly, substitution of Asp 281 with Ala resulted in a peptide with the greatest destabilization even though Ala possesses the largest helix propensity of the common 20 amino acids. Because this helix appears to be stabilized mainly by local electrostatics, we conclude that its structure is susceptible to even the most conservative mutations. These results provide support for the hypothesis that tumorigenic mutations induce local unfolding of p53. © 1999 John Wiley & Sons, Inc. Biopoly 49: 215–224, 1999     

Effect of DNA length and H4 acetylation on the thermal stability of reconstituted nucleosome particles

To examine the factors involved with nucleosome stability, we reconstituted nonacetylated particles containing various lengths (192, 162, and 152 base pairs) of DNA onto the Lytechinus variegatus nucleosome positioning sequence in the absence of linker histone. We characterized the particles and examined their thermal stability. DNA of less than chromatosome length (168 base pairs) produces particles with altered denaturation profiles, possibly caused by histone rearrangement in those core-like particles. We also examined the effects of tetra-acetylation of histone H4 on the thermal stability of reconstituted nucleosome particles. Tetra-acetylation of H4 reduces the nucleosome thermal stability by 0.8 degrees C as compared with nonacetylated particles. This difference is close to values published comparing bulk nonacetylated nucleosomes and core particles to ones enriched for core histone acetylation, suggesting that H4 acetylation has a dominant effect on nucleosome particle energetics.     

The Humicola grisea Cel12A enzyme structure at 1.2 A resolution and the impact of its free cysteine residues on thermal stability

As part of a program to discover improved glycoside hydrolase family 12 (GH 12) endoglucanases, we have extended our previous work on the structural and biochemical diversity of GH 12 homologs to include the most stable fungal GH 12 found, Humicola grisea Cel12A. The H. grisea enzyme was much more stable to irreversible thermal denaturation than the Trichoderma reesei enzyme. It had an apparent denaturation midpoint (T(m)) of 68.7 degrees C, 14.3 degrees C higher than the T. reesei enzyme. There are an additional three cysteines found in the H. grisea Cel12A enzyme. To determine their importance for thermal stability, we constructed three H. grisea Cel12A single mutants in which these cysteines were exchanged with the corresponding residues in the T. reesei enzyme. We also introduced these cysteine residues into the T. reesei enzyme. The thermal stability of these variants was determined. Substitutions at any of the three positions affected stability, with the largest effect seen in H. grisea C206P, which has a T(m) 9.1 degrees C lower than that of the wild type. The T. reesei cysteine variant that gave the largest increase in stability, with a T(m) 3.9 degrees C higher than wild type, was the P201C mutation, the converse of the destabilizing C206P mutation in H. grisea. To help rationalize the results, we have determined the crystal structure of the H. grisea enzyme and of the most stable T. reesei cysteine variant, P201C. The three cysteines in H. grisea Cel12A play an important role in the thermal stability of this protein, although they are not involved in a disulfide bond.     

Exploring subtype selectivity and metabolic stability of a novel series of ligands for the benzodiazepine binding site of the GABAA receptor

A novel series of agonists at the benzodiazepine binding site of the GABA_A receptor was prepared by functionalizing a known template. Adding substituents to the pyrazolone-oxygen of CGS-9896 led to a number of compounds with selectivities for either α2- or α1-containing GABA_A receptor subtypes offering an entry into indications such as anxiety and insomnia. In this communication, structure-activity relationship and efforts to increase in vitro stabilities are discussed.     

A conserved hydrophobic core at Bcl-xL mediates its structural stability and binding affinity with BH3-domain peptide of pro-apoptotic protein

Bcl-2 family proteins regulate apoptosis through their homo- and heterodimerization. By protein sequence analysis and structural comparison, we have identified a conserved hydrophobic core at the BH1 and BH2 domains of Bcl-2 family proteins. The hydrophobic core is stabilized by hydrophobic interactions among the residues of Trp137, Ile140, Trp181, Ile182, Trp188 and Phe191 in Bcl-x_L. Destabilization of the hydrophobic core can promote the protein unfolding and pore formation in synthetic lipid vesicles. Interestingly, though the hydrophobic core does not participate in binding with BH3 domain of pro-apoptotic proteins, disruption of the hydrophobic core can reduce the affinity of Bcl-x_L with BH3-domain peptide by changing the conformation of Bcl-x_L C-terminal residues that are involved in the peptide interaction. The BH3-domain peptide binding affinity and pore forming propensity of Bcl-x_L were correlated to its death-repressor activity, which provides new information to help study the regulatory mechanism of anti-apoptotic proteins. Meanwhile, as the tryptophans are conserved in the hydrophobic core, in vitro binding assay based on FRET of “Trp → AEDANS” can be devised to screen for new modulators targeting anti-apoptotic proteins as well as “multi-BH domains” pro-apoptotic proteins.     

A thermodynamic study of mesophilic, thermophilic, and hyperthermophilic L-arabinose isomerases: the effects of divalent metal ions on protein stability at elevated temperatures

To gain insight into the structural stability of homologous homo-tetrameric l-arabinose isomerases (AI), we have examined the isothermal guanidine hydrochloride (GdnHCl)-induced unfolding of AIs from mesophilic Bacillus halodurans (BHAI), thermophilic Geobacillus stearothermophilus (GSAI), and hyperthermophilic Thermotoga maritima (TMAI) using circular dichroism spectroscopy. The GdnHCl-induced unfolding of the AIs can be well described by a two-state reaction between native tetramers and unfolded monomers, which directly confirms the validity of the linear extrapolation method to obtain the intrinsic stabilities of these proteins. The resulting unfolding free energy (DeltaGU) values of the AIs as a function of temperature were fit to the Gibbs-Helmholtz equation to determine their thermodynamic parameters based on a two-state mechanism. Compared with the stability curves of BHAI in the presence and absence of Mn2+, those of holo GSAI and TMAI were more broadened than those of the apo enzymes at all temperatures, indicating increased melting temperatures (Tm) due to decreased heat capacity (DeltaGp). Moreover, the extent of difference in DeltaCp between the apo and holo thermophilic AIs is larger than that of BHAI. From these studies, we suggest that the metal dependence of the thermophilic AIs, resulting in the reduced DeltaCp, may play a significant role in structural stability compared to their mesophilic analogues, and that the extent of metal dependence of AI stability seems to be highly correlated to oligomerization.