Kinetic studies of sequence-specific binding of GCN4-bZIP peptides to DNA strands immobilized on a 27-MHz quartz-crystal microbalance.
Specific protein-DNA interaction was studied quantitatively by using a highly sensitive 27-MHz quartz-crystal microbalance (QCM). Biotinylated DNA double strands (21 bp, having a CRE site of 5'ATGACGTCAT3') were immobilized on an avidin-bound QCM surface, and sequence-specific binding of bZIP 56-mer peptides (having both the basic region for binding and the leucine zipper region for dimerization) to the DNA strand on the QCM was observed. The binding amount (Deltam) at the nanogram level and kinetic parameters such as association constants (Ka) and binding and dissociation rate constants (k1 and k-1) could be obtained from time courses of QCM frequency decreases. A bZIP peptide as a dimer was observed to bind sequence-specifically to one DNA strand having a CRE site. Ka values of ss-bZIP, in which the leucine-zipper region of bZIP was substituted by a Cys-Cys linkage, were largely decreased, and the sequence selectivity also disappeared. Ka values obtained by the QCM method showed good agreement with those obtained from the conventional gel mobility shift assay or from circular dichroism spectrum changes. When the specific sequence of the CRE site of DNA strands was partly changed, Ka values decreased by about a half due to the increase of the dissociation rate constant (k-1) independent of the binding rate constant (k1).
Okahata
Y
Y
Department of Biomolecular Engineering, Tokyo Institute of Technology, Yokohama, Japan. yokahta@bio.titech.ac.jp
Niikura
K
K
Sugiura
Y
Y
Sawada
M
M
Morii
T
T
D000595:Amino Acid Sequence; D001483:Base Sequence; D050976:Basic-Leucine Zipper Transcription Factors; D001665:Binding Sites; D015374:Biosensing Techniques; D002942:Circular Dichroism; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004271:DNA, Fungal / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D019281:Dimerization; D005656:Fungal Proteins / Q000378:metabolism*; D051790:G-Box Binding Factors; D007700:Kinetics; D008839:Microelectrodes; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D011791:Quartz; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors / Q000378:metabolism*
D050976:Basic-Leucine Zipper Transcription Factors; D017362:Cyclic AMP Response Element-Binding Protein; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D051790:G-Box Binding Factors; D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D011791:Quartz; D004247:DNA; D011494:Protein Kinases