Energetics of echinomycin binding to DNA

Differential scanning calorimetry and UV thermal denaturation have been used to determine a complete thermodynamic profile for the bis-intercalative interaction of the peptide antibiotic echinomycin with DNA. The new calorimetric data are consistent with all previously published binding data, and afford the most rigorous and direct determination of the binding enthalpy possible. For the association of echinomycin with DNA, we found ΔG° = –7.6 kcal mol^–1, ΔH = +3.8 kcal mol^–1 and ΔS = +38.9 cal mol^–1 K^–1 at 20°C. The binding reaction is clearly entropically driven, a hallmark of a process that is predominantly stabilized by hydrophobic interactions, though a deeper analysis of the free energy contributions suggests that direct molecular recognition between echinomycin and DNA, mediated by hydrogen bonding and van der Waals contacts, also plays an important role in stabilizing the complex.     

Energetics of DNA intercalation reactions

Isothermal titration calorimetry has been used to determine the binding enthalpy and heat capacity change (DeltaC(p)()) for a series of DNA intercalators, including ethidium, propidium, daunorubicin, and adriamycin. Temperature-dependent binding enthalpies were measured directly for the ligands, from which DeltaC(p)() values of -140 to -160 cal mol(-)(1) K(-)(1) were calculated. Published van't Hoff plots were reanalyzed to obtain DeltaC(p)() values of -337 to -423 cal mol(-)(1) K(-)(1) for the binding of actinomycin D to several DNA oligonucleotide duplexes with defined sequences. Heat capacity changes for DNA intercalation were found to correlate with the alterations in solvent-accessible surface area calculated from available high-resolution structural data. Multiple linear regression was used to derive the relationship DeltaC(p)() = 0. 382(+/-0.026)DeltaA(np) - 0.121(+/-0.077)DeltaA(p) cal mol(-)(1) K(-)(1), where DeltaA(np) and DeltaA(p) are the binding-induced changes in nonpolar and polar solvent-accessible surface areas (in square angstroms), respectively. The DeltaC(p)() terms were used to estimate the hydrophobic contribution to intercalative binding free energies, yielding values that ranged from -11.2 (ethidium) to -30 kcal mol(-)(1) (actinomycin D). An attempt was made to parse the observed binding free energies of ethidium and propidium into five underlying contributions. Such analysis showed that the DNA binding behavior of these simple intercalators is driven almost equally by hydrophobic effects and van der Waals contacts within the intercalation site.     

Anthracycline antibiotics. Interaction with DNA and nucleosomes and inhibition of DNA synthesis

We report studies of the interaction of four anthracycline antibiotics, iremycin (IM), daunomycin (DM), aclacinomycin A (AM), and violamycin B1 (VM), with naked DNA, nucleosomal core particles, and 175 base pair (bp) nucleosomes lacking histone H1. In all cases the binding strength increases in the order IM less than DM approximately AM less than VM. The binding substrates increased in affinity for the drugs in the following order: core particles less than 175-bp nucleosomes less than DNA. The apparent DNA length increment per drug bound decreases in the progression IM greater than DM greater than AM greater than VM, the same serial order as is characterized by increasing binding affinity. Dichroism amplitude measurements show that for all drugs the long-wavelength absorbance transition moment is tilted by 26-29 degrees relative to the plane perpendicular to the helix axis; this angle probably corresponds to the long axis tilt of the intercalated chromophore. Finally, it was found that the ability of the drugs to inhibit DNA synthesis by Escherichia coli DNA polymerase I increases in the same order as their binding affinity.     

Sequence- and structural-selective nucleic acid binding revealed by the melting of mixtures

A simple method for the detection of sequence- and structural-selective ligand binding to nucleic acids is described. The method is based on the commonly used thermal denaturation method in which ligand binding is registered as an elevation in the nucleic acid melting temperature (T_m). The method can be extended to yield a new, higher -throughput, assay by the simple expediency of melting designed mixtures of polynucleotides (or oligonucleotides) with different sequences or structures of interest. Upon addition of ligand to such mixtures at low molar ratios, the T_m is shifted only for the nucleic acid containing the preferred sequence or structure. Proof of principle of the assay is provided using first a mixture of polynucleotides with different sequences and, second, with a mixture containing DNA, RNA and two types of DNA:RNA hybrid structures. Netropsin, ethidium, daunorubicin and actinomycin, ligands with known sequence preferences, were used to illustrate the method. The applicability of the approach to oligonucleotide systems is illustrated by the use of simple ternary and binary mixtures of defined sequence deoxyoligonucleotides challenged by the bisanthracycline WP631. The simple mixtures described here provide proof of principle of the assay and pave the way for the development of more sophisticated mixtures for rapidly screening the selectivity of new nucleic acid binding compounds.     

Calorimetry outside the box: a new window into the plasma proteome

Differential scanning calorimetry provides a new window into the plasma proteome. Plasma from normal individuals yields a characteristic, reproducible thermogram that appears to represent the weighted sum of denaturation profiles of the most abundant constituent plasma proteins. Plasma from diseased individuals yields dramatically different signature thermograms. Thermograms from individuals suffering from rheumatoid arthritis, systemic lupus, and Lyme disease were measured. Each disease appears to have a distinctive and characteristic thermogram. The difference in thermograms between normal and diseased individuals is not caused by radical changes in the concentrations of the most abundant plasma proteins but rather appears to result from interaction of as yet unknown biomarkers with the major plasma proteins. These results signal a novel use for calorimetry as a diagnostic tool.     

Calorimetric analysis of the plasma proteome: Identification of type 1 diabetes patients with early renal function decline

Background

Microalbuminuria (MA) has been questioned as a predictor of progressive renal dysfunction in patients with type 1 diabetes (T1D). Consequently, new clinical end points are needed that identify or predict patients that are at risk for early renal function decline (ERFD). The potential clinical utility of differential scanning calorimetry (DSC) analysis of blood plasma and other biofluids has recently been reported. This method provides an alternate physical basis with which to study disease-associated changes in the bulk plasma proteome.

Methods

DSC analysis of blood plasma was applied to identify unique signatures of ERFD in subjects enrolled in the 1st Joslin Study of the Natural History of Microalbuminuria in Type 1 Diabetes, a prospective cohort study of T1D patients. Recent data suggests that differences in the plasma peptidome of these patients correlate with longitudinal measures of renal function. Differences in DSC profile (thermogram) features were evaluated between T1D MA individuals exhibiting ERFD (n = 15) and matched control subjects (n = 14).

Results

The average control group thermogram resembled a previously defined healthy thermogram. Differences were evident between ERFD and control individuals. Heat capacity values of the main two transitions were found to be significant discriminators of patient status.

Conclusions

Results from this pilot study suggest the potential utility of DSC proteome analysis to prognostic indicators of renal disease in T1D.

General significance

DSC shows sensitivity to changes in the bulk plasma proteome that correlate with clinical status in T1D providing additional support for the utility of DSC profiling in clinical diagnostics.     

An Improved Model for the hTERT Promoter Quadruplex

Mutations occur at four specific sites in the hTERT promoter in >75% of glioblastomas and melanomas, but the mechanism by which the mutations affect gene expression remains unexplained. We report biophysical computational studies that show that the hTERT promoter sequence forms a novel G-quadruplex structure consisting of three contiguous, stacked parallel quadruplexes. The reported hTERT mutations map to the central quadruplex within this structure, and lead to an alteration of its hydrodynamic properties and stability.     

Structure of the Pseudorabies Virus Capsid: Comparison with Herpes Simplex Virus Type 1 and Differential Binding of Essential Minor Proteins

The structure of pseudorabies virus (PRV) capsids isolated from the nucleus of infected cells and from PRV virions was determined by cryo-electron microscopy (cryo-EM) and compared to herpes simplex virus type 1 (HSV-1) capsids. PRV capsid structures closely resemble those of HSV-1, including distribution of the capsid vertex specific component (CVSC) of HSV-1, which is a heterodimer of the pUL17 and pUL25 proteins. Occupancy of CVSC on all PRV capsids is near 100%, compared to ~ 50% reported for HSV-1 C-capsids and 25% or less that we measure for HSV-1 A- and B-capsids. A PRV mutant lacking pUL25 does not produce C-capsids and lacks visible CVSC density in the cryo-EM-based reconstruction. A reconstruction of PRV capsids in which green fluorescent protein was fused within the N-terminus of pUL25 confirmed previous studies with a similar HSV-1 capsid mutant localizing pUL25 to the CVSC density region that is distal to the penton. However, comparison of the CVSC density in a 9-Å-resolution PRV C-capsid map with the available crystal structure of HSV-1 pUL25 failed to find a satisfactory fit, suggesting either a different fold for PRV pUL25 or a capsid-bound conformation for pUL25 that does not match the X-ray model determined from protein crystallized in solution. The PRV capsid imaged within virions closely resembles C-capsids with the addition of weak but significant density shrouding the pentons that we attribute to tegument proteins. Our results demonstrate significant structure conservation between the PRV and HSV capsids.