High mobility group protein (HMG) is known to be involved in the formation of high order structure of chromatin. HMGs with minor-groove binding ability in the AT-rich DNA region play a vital role in controlling gene transcription activity. In this report, a 18-residue HMG-I/DAT1 chimeric peptide, PRGRPKGKTLREPRGRPY, was designed and synthesized containing two repetitive PRGRP units and a linking peptide, KGKTLRE, as a targeted DNA-binding peptide. The segment PRGRP is derived from HMG-1 while KGKTLRE is from the DAT1 peptide. Using gel-mobility shift assay and 32P-end labeled 27 bp AT-rich DNA, the dissociation constant of this chimeric peptide was found to he 4.7 × 10^(-6) M, that is, l0^4 times stronger than that of the PRGRP segment stand alone (> 10^(-2) M). In addition, the binding constant was found to increase with the length of AT-rich DNA. The possible DNA binding site of the HMG-I/DAT1 chimeric peptide is determined by footprinting experiments using a minor-groove cleaving agent ruthenium(Ⅲ)-Schiff base complex and a 135-bp 32P-5'-end-labeled DNA restriction fragment of Hind Ⅲ/Rsa I from plasmid pBR322 DNA. The major pattern protected by the HMG-I/DAT1 chimeric peptide exhibits a preference for 5'-AAAT-3' of the AT-rich region. Therefore, this novel design HMG-I/DAT1 chimeric peptide possesses not only a high affinity to AT-rich DNA but also the sequence-specific binding in the minor groove of DNA, which may further lead to the design of short synthetic peptides for therapeutic applications
Relation:
Journal of the Chinese Chemical Society=中國化學會會誌 45(5), pp.619-624