Myb-DNA Recognition: Role of Tryptophan Residues and Structural Changes of the Minimal DNA Binding Domain of c-Myb
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- 作者：Loussiné ; e Zargarian ; Vé ; ronique Le Tilly ; Nadè ; ge Jamin ; Alain Chaffotte ; Odd S. Gabrielsen ; Flavio Toma ; and Bernard Alpert
- 刊名：Biochemistry
- 出版年：1999
- 出版时间：February 9, 1999
- 年：1999
- 卷：38
- 期：6
- 页码：1921 - 1929
- 全文大小：200K
- 年卷期：v.38,no.6(February 9, 1999)
- ISSN：1520-4995
文摘
The Myb oncoprotein specifically binds DNA by a domain composed of three imperfect repeats,R1, R2, and R3, each containing 3 tryptophans. The tryptophan fluorescence of the minimal binding domain,R2R3, of c-Myb was used to monitor structural flexibility changes occurring upon DNA binding to R2R3.The quenching of the Trp fluorescence by DNA titration shows that four out of the six tryptophans areinvolved in the formation of the specific R2R3-DNA complex and the environment of the tryptophanresidues becomes more hydrophobic in the complex. The fluorescence intensity quenching of thetryptophans by binding of R2R3 to DNA is consistent with the decrease of the decay time: 1.46 ns forfree R2R3 to 0.71 ns for the complexed protein. In the free R2R3, the six tryptophans are equally accessibleto the iodide and acrylamide quenchers with a high collisional rate constant (4 × 109 and 3 × 109 M-1s-1, respectively), indicating that R2R3 in solution is very flexible. In the R2R3-DNA complex, no Trpfluorescence quenching is observed with iodide whereas all tryptophan residues remain accessible toacrylamide with a collisional rate constant slightly slower than that in the free state. These results indicatethat (i) a protein structural change occurs and (ii) the R2R3 molecule keeps a high mobility in thecomplex.The complex formation presents a two-step kinetics: a fast step corresponding to the R2R3-DNA association (7 × 105 M-1 s-1) and a slower one (0.004 s-1), which should correspond to a structuralreorganization of the protein including a reordering of the water molecules at the protein-DNA interface.