Enthalpy-driven nuclease-like activity and mechanism of peptide-chlorambucil conjugates.

We report the results of attaching the anticancer drug chlorambucil (CLB) to two high-affinity DNA binding peptides: Met-Hyp-Arg-Lys-(Py)4-Lys-Arg-NH2 (HyM-10) and Gln-Hyp-Arg-Lys-(Py)4-Lys-Arg-NH2 (HyQ-10). These CLB-peptide conjugates cleave DNA very effectively and sequence-selectively without the use of chemicals, heat, or UV irradiation. Polyacrylamide gel electrophoresis identifies the sites where CLB-HyM-10 and CLB-HyQ-10 attack a complementary pair of 5'-(32)P-labeled duplexes derived from pBR322 in the absence of piperidine or other chemical additives. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) has confirmed the preferential cleavage sites as well as a novel stepwise cleavage mechanism of sequence-selective DNA cleavage. Resembling restriction endonucleases, the CLB-peptide conjugates appear to be capable of producing double strand DNA breaks. Circular dichroism studies show that 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. 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.

Detection of multiple network-based allosteric interactions between peptides and arrays of DNA binding sites.

Quantitative DNase I footprinting shows that three designed peptides containing N-methylpyrrole (Py) moieties display different types of network-based allosteric communication in binding to DNA: circuit type, incomplete-circuit type, and non-circuit type characterized by interstrand bidentate interactions. Positive cooperative binding of all three peptides to individual DNA binding sites is commonly observed. CD spectral characterization of the interaction between peptides and model undecanucleotide duplexes is consistent with the footprinting results and supports the allosteric model. This study provides insights relating to the interaction network nature of allostery in complex DNA-small molecule interactions.