The oncogene product c-myb is a transcriptional modulator and is known
to play important roles in cell growth and differentiation. It binds
to DNA in a sequence specific manner and its cognate sequence motifs h
ave been detected in the genes of proteins implying its role in a vari
ety of regulatory functions. The protein has a DNA binding domain cons
isting of three imperfect repeats with highly conserved tryptophans at
regular spacings in each of the repeats. We have carried out a variet
y of investigations on the structure and interactions of the DNA bindi
ng domain of Drosophila c-myb and its cognate DNA target sequences. Th
e domain has been bacterially over-expressed by subcloning a segment o
f the gene coding for the domain in a pET 11d vector and transforming
it into E. coli BL21 (DE3). Circular dichroism of the protein has reve
aled that the domain is largely helical in nature. Fluorescence invest
igations indicated that three out of the nine tryptophans are solvent
exposed and the others are buried in the interior. The recombinant pro
tein is able to distinguish between specific and non-specific DNA targ
ets in its binding and the interaction is largely electrostatic in nat
ure in both cases. Dynamic fluorescence quenching experiments suggeste
d that the DNA binding sites on the protein for specific and non-speci
fic DNA targets are physically different. Most of the conserved trypto
phans are associated with the specific DNA binding site. Simulated ann
ealing and molecular dynamic simulations in a water matrix have been u
sed to predict an energetically favoured conformation for the protein.
Calculation of surface accessibilities of the individual residues sho
ws that nearly 60% of the residues are less than 50% accessible to the
solvent. Two and three dimensional NMR experiments with isotopically
labelled protein have enabled spin system identification for many resi
due type and the types of residues involved in hydrophobic core format
ion in the protein. In an attempt to see the DNA surface possibly invo
lved in specific interaction with the protein, a three-dimensional str
ucture of a 12 mer cognate DNA has been determined by NMR in conjuncti
on with restrained energy minimization. The recognition sequence shows
interesting structural characteristics that may have important roles
in specific interaction. (C) 1997 Elsevier Science B.V.