ABSTRACTcis-Diamminedichloroplatinum(II) (cisplatin) is a widely used anticancer drug that binds to and crosslinks DNA. The major DNA adduct of the drug results from coordination of two adjacent guanine bases to platinum to form the intrastrand crosslink cis-[Pt(NH3)2{d(GpG)-N7(1), -N7(2)}] (cis-Pt-GG). In the present study, spectroscopic and calorimetric techniques were employed to characterize the influence of this crosslink on the conformation, thermal stability, and energetics of a site-specifically platinated 20-mer DNA duplex. CD spectroscopic and thermal denaturation data revealed that the crosslink alters the structure of the host duplex, consistent with a shift from a B-like to an A-like conformation; lowers its thermal stability by -9°C; and reduces its thermodynamic stability by 6.3 kcal/mol at 25°C, most of which is enthalpic in origin; but it does not alter the two-state melting behavior exhibited by the parent, unmodified duplex, despite the significant crosslinkinduced changes noted above. The energetic consequences of the cis-Pt-GG crosslink are discussed in relation to the structural perturbations it induces in DNA and to how these crosslinkinduced perturbations might modulate protein binding.The chemotherapeutic efficacy of the anticancer drug cisplatin is derived from its ability to bind and crosslink DNA, the major adduct being the cis-Pt-GG intrastrand crosslink (1). Previous crystallographic, NMR, and gel electrophoretic investigations have evaluated the impact of this crosslink on the structure and conformation of the host duplex (1-9). These studies indicate that formation of the cis-Pt-GG crosslink unwinds DNA by 130and bends it by 34-55°in the direction of the major groove. Avery recent crystallographic study revealed that the crosslink can induce formation of an A-form structural subdomain, thereby creating a hybrid DNA duplex with an A-B junction (8). Such cis-Pt-GG-induced alterations in duplex structure have been implicated in the promotion of specific interactions with cellular proteins that contain one or more high mobility group domainsfor example, HMG1, Ixrl, and human upstream-binding factor (hUBF) (8,(10)(11)(12)(13)(14)(15). When bound by such cellular proteins, the cis-Pt-GG sites are shielded from excision repair (11,13,16,17), thereby enhancing the cytotoxic properties of the drug. Unlike the detailed structural information about the major cisplatin-DNA adduct, comparatively little is known about its corresponding energetic consequences. Such thermodynamic data would reveal how the crosslink influences duplex stability, a property that has been implicated in the modulation of protein recognition and binding (18)(19)(20). To address this deficiency, we have used a combination of calorimetric and spectroscopic techniques to characterize the effect of a single cis-Pt-GG intrastrand crosslink on the conformation, thermal stability, and energetics of a 20-mer DNA duplex. The sequences of unmodified (GG20) and modified (cis-Pt-GG20) duplexes examined are depicted in Fig. 1. Inve...

Thermal denaturation of equinatoxin II (EqTxII) in glycine buffer solutions (pH 1.1, 2.0, 3.0, and 3.5) and in triple distilled water (pH 5.5-6.0) was examined by differential scanning calorimetry, UV and CD spectroscopy and fluorescence emission spectroscopy of the added hydrophobic fluorescent probe ANS. At pH 5.5-6.0 and at temperatures below 60 degrees C, the protein exists in a native state characterized by a pronounced tertiary structure, a beta-rich secondary structure and a low degree of ANS-binding. At higher temperatures, it undergoes a two-state conformational transition, (delta H degree)VH = (delta H degree)DSC, into an unfolded state, which is characterized by a complete collapse of its tertiary structure and an incomplete denaturation of its secondary structure. At acidic pH, the EqTxII temperature-induced conformational transition appears at lower temperatures as non-two-state transition accompanied by the formation of an intermediate state which shows characteristics of molten globules, i.e., absence of defined tertiary structure, increase in alpha-rich secondary structure, and high affinity for ANS. At pH 2.0, the low-temperature initial state of EqTxII is already partially denatured; the tertiary structure is partially disrupted, and a pronounced inequality (delta H degree)VH > (delta H degree)DSC is observed. At pH value of 1.1 and below 60 degrees C, EqTxII exists in a stable acid-denatured compact state which shows all the characteristics of a molten globule, which even at 95 degrees C is not completely denatured. According to numerous studies on the pore forming toxins, such acid-denatured compact states may contribute to the protein's ability to penetrate into biological membranes.

Hairpin polyamides are synthetic ligands for sequence-specific recognition in the minor groove of doublehelical DNA. A thermodynamic characterization of the DNAbinding properties exhibited by a six-ring hairpin polyamide, ImPyPy-y-PyPyPy-P-Dp (where Im = imidazole, Py = pyrrole, y = -y-aminobutyric acid, ,B = ,I-alanine, and Dp = dimethylaminopropylamide), reveals an -1-2 kcal/mol greater affinity for the designated match site, 5'-TGTTA-3', relative to the single base pair mismatch sites, 5'-TGGTA-3' and 5'-TATTA-3'. The enthalpy and entropy data at 200C reveal this sequence specificity to be entirely enthalpic in origin. Correlations between the thermodynamic driving forces underlying the sequence specificity exhibited by ImPyPy-y-PyPyPy-f3-Dp and the structural properties of the heterodimeric complex of PyPyPy and ImPyPy bound to the minor groove of DNA provide insight into the molecular forces that govern the affinity and specificity of pyrrole-imidazole polyamides.

Crescent-shaped synthetic ligands containing aromatic amino acids have been designed for specific recognition of predetermined DNA sequences in the minor groove of DNA. Simple rules have been developed that relate the side-by-side pairings of Imidazole (Im) and Pyrrole (Py) amino acids to their predicted target DNA sequences. We report here thermodynamic characterization of the DNA-binding properties of the six-ring hairpin polyamide, ImImPy-gamma-PyPyPy-beta-Dp (where gamma = gamma-aminobutyric acid, beta = beta-alanine, and Dp = dimethylaminopropylamide). Our data reveal that, at 20 degrees C, this ligand binds with a relatively modest 1.8-fold preference for the designated match site, 5'-TGGTA-3', over the single base pair mismatch site, 5'-TGTTA-3'. By contrast, we find that the ligand exhibits a 102-fold greater affinity for its designated match site relative to the double base pair mismatch site, 5'-TATTA-3'. These results demonstrate that the energetic cost of binding to a double mismatch site is not necessarily equal to twice the energetic cost of binding to a single mismatch site. Our calorimetrically measured binding enthalpies and calculated entropy data at 20 degrees C reveal the ligand sequence specificity to be enthalpic in origin. We have compared the DNA-binding properties of ImImPy-gamma-PyPyPy-beta-Dp with the hairpin polyamide, ImPyPy-gamma-PyPyPy-beta-Dp (an Im --> Py "mutant"). Our data reveal that both ligands exhibit high affinities for their designated match sites, consistent with the Dervan pairing rules. Our data also reveal that, relative to their corresponding single mismatch sites, ImImPy-gamma-PyPyPy-beta-Dp is less selective than ImPyPy-gamma-PyPyPy-beta-Dp for its designated match site. This result suggests, at least in this case, that enhanced binding affinity can be accompanied by some loss in sequence specificity. Such systematic comparative studies allow us to begin to establish the thermodynamic database required for the rational design of synthetic polyamides with predictable DNA-binding affinities and specificities.