Characterization of the DNA binding properties of the bHLH domain of Deadpan to single and tandem sites.
The basic helix-loop-helix domain of the Drosophila transcription factor Deadpan (Dpn) was prepared by total chemical protein synthesis in order to characterize its DNA binding properties. Circular dichroism spectroscopy was used to correlate structural changes in Dpn with physiologically relevant monovalent (KCl) and divalent (MgCl2) cation concentrations. In addition, we have used electrophoretic mobility shift assay (EMSA) and fluorescence anisotropy methods to determine equilibrium dissociation constants for the interaction of Dpn with two biologically relevant promoters involved in neural development and sex determination pathways. In this study, we have optimized DNA binding conditions for Dpn, and we have found a markedly higher DNA binding affinity for Dpn than reported for other bHLH domain transcription factors. Dpn binds as a homodimer (Kd = 2.6 nM) to double-stranded oligonucleotides containing the binding site CACGCG. In addition, we found that Dpn bound with the same affinity to a single or tandem binding site, indicating no cooperativity between adjacent DNA-bound Dpn dimers. DNA binding was also monitored as a function of physiologically relevant KCl and MgCl2 concentrations, and we found that this activity was significantly different in the presence and absence of the nonspecific competitor poly(dI-dC). Moreover, Dpn displayed moderate sequence selectivity, exhibiting a 100-fold higher binding affinity for specific DNA than for poly(dI-dC). This study constitutes the first detailed biophysical characterization of the DNA binding properties of a bHLH protein.
Winston
R L
RL
Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA.
Millar
D P
DP
Gottesfeld
J M
JM
Kent
S B
SB
D000595:Amino Acid Sequence; D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins; D001665:Binding Sites; D002942:Circular Dichroism; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D004330:Drosophila; D029721:Drosophila Proteins; D005454:Fluorescence Polarization; D018257:Helix-Loop-Helix Motifs; D007700:Kinetics; D015636:Magnesium Chloride / Q000737:chemistry; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism*; D011189:Potassium Chloride / Q000737:chemistry; D011485:Protein Binding; D020449:Repetitive Sequences, Amino Acid; D013050:Spectrometry, Fluorescence; D014461:Ultracentrifugation
D051792:Basic Helix-Loop-Helix Proteins; D004247:DNA; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D015636:Magnesium Chloride; D009687:Nuclear Proteins; D010446:Peptide Fragments; D011189:Potassium Chloride; C078020:Dpn protein, Drosophila