Aromatic amino acids are critical for stability of the bicoid homeodomain.
The Drosophila Bicoid (Bcd) protein plays a dual role as a transcription and 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 native state but is dramatically enhanced ( approximately 20-fold) upon denaturation. Similar results were obtained with the Ultrabithorax HD (Ubx-HD), suggesting that the unusual tryptophan fluorescence may be a general phenomenon of HD proteins. We have used site-directed mutagenesis to explore the role of aromatic acids in the structure of the Bcd-HD and to evaluate the proposal that interactions between the strictly conserved Trp residue in HDs and nearby aromatic residues are responsible for the fluorescence quenching in the native state. We determined that both Trp-48 and Phe-8 in the N-terminal region of the HD are individually necessary for structural stability of the Bcd-HD, the latter most likely as a factor coordinating the orientation of the N-terminal helix I and the recognition helix for efficient binding to a DNA target.
Subramaniam
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V
Department of Molecular Biology, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, D-37077 Göttingen, Germany.
Jovin
T M
TM
Rivera-Pomar
R V
RV
D019943:Amino Acid Substitution; D000598:Amino Acids, Cyclic / Q000737:chemistry*; D000818:Animals; D001665:Binding Sites; D002942:Circular Dichroism; D004330:Drosophila; D029721:Drosophila Proteins; D018398:Homeodomain Proteins / Q000737:chemistry* / Q000378:metabolism*; D006358:Hot Temperature; D019476:Insect Proteins / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D015534:Trans-Activators / Q000737:chemistry* / Q000378:metabolism*; D014364:Tryptophan
D000598:Amino Acids, Cyclic; D029721:Drosophila Proteins; D018398:Homeodomain Proteins; D019476:Insect Proteins; D011994:Recombinant Proteins; D015534:Trans-Activators; C056478:bcd protein, Drosophila; D014364:Tryptophan