Determination of the dissociation constants for recombinant c-Myc, Max, and DNA complexes: the inhibitory effect of linoleic acid on the DNA-binding step.
c-Myc, the protein product of protooncogene c-myc, functions in cell proliferation, differentiation, and neoplastic disease. In this study, recombinant c-Myc and Max proteins, encompassing DNA binding (basic region) and dimerization (helix-loop-helix/leucine zipper) domain of human origin, were expressed in bacteria as Myc87 and Max85. Myc87 was purified under denatured conditions and was renatured again. The dissociation constant for the protein dimers and for dimer/DNA complexes were not detectable by isothermal titration calorimetry because of the low degree of solubility of Myc87 and Max85. Therefore, we set up equations which were used to determine the dissociation constants from the proportion of protein-DNA complexes. The dimer dissociation constants in TBS were 5.90(+/-0.54)x10(-7)M for Max85/Max85 homodimer, 6.85(+/-0.25)x10(-3)M for Myc87/Myc87 homodimer, and 2.55(+/-0.29)x10(-8)M for Myc87/Max85 heterodimer, and the DNA-binding dissociation constants in TBS were 1.33(+/-0.21)x10(-9)M for Max85/Max85/DNA, 2.27(+/-0.08)x10(-12)M for Myc87/Myc87/DNA, and 4.43(+/-0.37)x10(-10)M for Myc87/Max85/DNA. In addition, we revealed that linoleic acid which is known as an inhibitor for the formation of Max/Max/DNA complex reduced the affinity of Max homodimer for DNA. This result indicates that linoleic acid may bind to the DNA-binding region of Max homodimer.
Department of Chemistry, Seoul National University, Seoul, Republic of Korea.
Kyung Chae
KC
Jung
Ho Sung
HS
Rhee
Chi Hoon
CH
Park
Chul-Hak
CH
Yang
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D001665:Binding Sites; D003198:Computer Simulation; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000737:chemistry*; D004586:Electrophoresis; D007700:Kinetics; D019787:Linoleic Acid / Q000737:chemistry*; D046911:Macromolecular Substances / Q000737:chemistry; D008956:Models, Chemical; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry; D012097:Repressor Proteins / Q000737:chemistry*; D014157:Transcription Factors / Q000737:chemistry*
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D004268:DNA-Binding Proteins; C106465:MNT protein, human; C116115:MYCBP protein, human; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D004247:DNA; D019787:Linoleic Acid