Myb-DNA recognition: Role of tryptophan residues and structural changes ofthe minimal DNA binding domain of c-Myb

The Myb oncoprotein specifically binds DNA by a domain composed of three im perfect repeats, R-1, R-2, and R-3, each containing 3 tryptophans. The tryp tophan fluorescence of the minimal binding domain, R2R3, of c-Myb was used to monitor structural flexibility changes occurring upon DNA binding to R2R 3 The quenching of the Trp fluorescence by DNA titration shows that four ou t of the six tryptophans are involved in the formation of the specific R2R3 -DNA complex and the environment of the tryptophan residues becomes more hy drophobic in the complex. The fluorescence intensity quenching of the trypt ophans by binding of R2R3 to DNA is consistent with the decrease of the dec ay time: 1.46 ns for free R2R3 to 0.71 ns for the complexed protein. In the free R2R3, the six tryptophans are equally accessible to the iodide and ac rylamide quenchers with a high collisional rate constant (4 x 10(9) and 3 x 10(9) M-1 s(-1), respectively), indicating that R2R3 in solution is very f lexible. In the R2R3-DNA complex, no Trp fluorescence quenching is observed with iodide whereas all tryptophan residues remain accessible to acrylamid e with a collisional rate constant slightly slower than that in the free st ate. These results indicate that (i) a protein structural change occurs and (ii) the R2R3 molecule keeps a high mobility in the complex. The complex f ormation presents a two-step kinetics: a fast step corresponding to the R2R 3-DNA association (7 x 10(5) M-1 s(-1)) and a slower one (0.004 s(-1)), whi ch should correspond to a structural reorganization of the protein includin g a reordering of the water molecules at the protein-DNA interface.