Thermal stability of the DNA-binding domain of the Myb oncoprotein.
The DNA-binding domain of the c-myb protooncogene product consists of three homologous tandem repeats of 51-52 amino acids (denoted as R1, R2, and R3 from the N-terminal side). In order to analyze conformational and thermodynamic characteristics of the homologous repeats, we have examined the DNA-binding domain by circular dichroism (CD) and differential scanning calorimetry (DSC). The CD spectra for the three individual repeats are significantly different in the fine profiles, indicating subtle differences in their conformations. The melting analyses for the fragments show that the thermal stability of each fragment is different from one another, with the following order of stability: R1(Tm = 61 degrees C) approximately greater than R3(57 degrees C) >> R2(43 degrees C), where R2 is much less stable than the other repeats. The denaturing process for the whole DNA-binding domain, measured by DSC, is characterized by a very broad transition ranging from 30 to 80 degrees C. The denaturation curve can be fit well by a three-state transition with one intermediate state. The transition temperature for the native-to-intermediate transition coincides with the melting temperature of R2, indicating that the intermediate state corresponds to the unfolding of unstable R2. The CD spectrum of the whole domain is almost identical to the sum of the individual spectra. Thus, these results suggest that the individual repeats in the whole DNA-binding domain behave independently in terms of conformation and stability. The addition of DNA to the DNA-binding fragment drastically changed the melting profile, in which the broad transition curve was replaced by a sharp peak at 58 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)
Sarai
A
A
Life Science Center, Institute of Chemical & Physical Research (RIKEN), Ibaraki, Japan.
Uedaira
H
H
Morii
H
H
Yasukawa
T
T
Ogata
K
K
Nishimura
Y
Y
Ishii
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D002152:Calorimetry, Differential Scanning; D002942:Circular Dichroism; D004247:DNA; D004268:DNA-Binding Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011489:Protein Denaturation; D011518:Proto-Oncogene Proteins / Q000737:chemistry*; D020598:Proto-Oncogene Proteins c-myb; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics
D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; D020598:Proto-Oncogene Proteins c-myb; D004247:DNA