Aromatic amino acids are critical for stability of the bicoid homeodomain

The Drosophila Bicoid (Bcd) protein plays a dual role as a transcription an d translation factor dependent on the unique DNA and RNA binding properties of the homeodomain (HD). We have used circular dichroism and fluorescence spectroscopy to probe the structure and stability of the Bcd-HD, for which a high resolution structure is not yet available. The fluorescence from the single tryptophan residue in the HD (Trp-48) is strongly quenched in the n ative state but is dramatically enhanced (similar to 20-fold) upon denatura tion. Similar results were obtained with the Ultrabithorax HD (Ubx-HD), sug gesting that the unusual tryptophan fluorescence may be a general phenomeno n of HD proteins. We have used site-directed mutagenesis to explore the rol e of aromatic acids in the structure of the Bcd-HD and to evaluate the prop osal that interactions between the strictly conserved Trp residue in HDs an d nearby aromatic residues are responsible for the fluorescence quenching i n the native state. We determined that both Trp-48 and Phe-8 in the N-termi nal region of the HD are individually necessary for structural stability of the Bcd-HD, the latter most likely as a factor coordinating the orientatio n of the N-terminal helix I and the recognition helix for efficient binding to a DNA target.