| Summary: | 博士 === 東海大學 === 化學系 === 102 === The first part of this thesis reports the study of the energetic basis of complex DNA–peptide interactions relating to allosteric interactions. In common with other designed peptides, ten new conjugates incorporating the XPRK or XHypRK motif (Hyp = hydroxyproline) attached to a N-methylpyrrole (Py) tract with a basic tail have been found to display cooperative binding to DNA involving multiple monodentate as well as interstrand bidentate interactions. Quantitative DNase I footprinting show that allosteric communication via cooperative binding to multiple sites on complementary DNA strands corresponds to two different types of DNA–peptide interaction network. Temperature variation experiments using a dodecapeptide RY-12 show that lower temperature (25 °C) favors a circuit type of allosteric interaction network, whereas higher temperatures (31 and 37 °C) afford only a partial-circuit type of network. Circular dichroism studies show that the peptides induce significant local conformational changes in DNA via the minor groove, with apparently dimeric binding stoichiometry.
Isothermal titration calorimetry reveals that the peptides are strongly exothermic upon binding to a model 13-mer DNA duplex, as characterized by ΔH ranging from −14.7 to −74.4 kcal mol−1, and also high TΔS ranging from −6.5 to −65.9 kcal mol−1. Distinctive enthalpy–entropy compensation (EEC) relationships are demonstrated for the interaction of all twelve designed peptides with DNA, affording a straight line of slope close to unity when ΔH is plotted versus TΔS, with a y-axis intercept (average ΔG) corresponding to −8.5 kcal mol−1, while the observed ΔG ranges from −8.2 to −9.1 kcal mol−1 for the peptides. The EEC seen with peptide RY-12 binding to the model duplex persists throughout various incubation temperatures. The net compensation of energy between the favorable negative ΔH and unfavorable negative ΔS components thus constrains the value of net binding free energy ΔG within a remarkably constant range, as is clearly visible in a 3-dimensional energetic plot.
For the second part of this thesis, both polyacrylamide gel electrophoresis and MALDI-TOF experiments showed that chlorambucil-peptides conjugates CLB-HyM-10 and CLB-HyQ-10 can readily cleave DNA duplexes very effectively and sequence-selectively in the absence of heat, chemical additives and UV irradiation.
Circular dichroism studies show that the conjugates CLB–HyM-10 and CLB–HyQ-10 induce significant local conformational changes in DNA via the minor groove, possibly with dimeric binding stoichiometry. The energetic basis of DNA binding by these conjugates has been investigated by isothermal titration calorimetry, revealing that the binding of both the peptides and their CLB conjugates is overwhelmingly enthalpy-driven.
The maintenance of a conserved negative binding free energy in DNA–conjugate interactions is a crucial feature of the universal enthalpy–entropy compensation phenomenon. It is concluded that the strongly enthalpy-driven binding of CLB–peptide conjugates to preferred loci in DNA furnishes the required proximity effect to generate the observed nuclease-like sequence-selective cleavage.
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