Gadhavi P, Morgan P J, Alefounder P, Harding S E
Department of Biochemistry, University of Cambridge, UK.
Eur Biophys J. 1996;24(6):405-12. doi: 10.1007/BF00576712.
It has previously been suggested that the DNA binding domain (residues 1 to 147) of the yeast transcriptional activator GAL4 exists in solution in dimeric form, with the region responsible for dimerisation somewhere between residues 74 and 147. In this study limited proteolysis and carboxy-terminal deletions of the DNA binding domain (residues 1 to 147) of the yeast transcriptional activator GAL4 followed by subsequent characterization by equilibrium sedimentation in the analytical ultracentrifuge have been used to define more precisely the regions required for DNA binding and protein self-association. Sedimentation equilibrium analyses confirmed that the 'hydrophobic region' of the protein (residues 54-97, which contains a larger proportion of alpha-helix), is essential for dimerisation, with an apparent dissociation constant K(D,app), of approximately 50 microM for the 1-94 residue peptide and approximately 20 microM for the 1-147 residue peptide. Our studies do not rule out the possible formation of small amounts of additional higher order complexes.
先前有研究表明，酵母转录激活因子GAL4的DNA结合结构域（第1至147位氨基酸残基）在溶液中以二聚体形式存在，负责二聚化的区域位于第74至147位氨基酸残基之间。在本研究中，通过对酵母转录激活因子GAL4的DNA结合结构域（第1至147位氨基酸残基）进行有限蛋白酶解和羧基末端缺失，随后在分析型超速离心机中通过平衡沉降进行表征，以更精确地确定DNA结合和蛋白质自缔合所需的区域。沉降平衡分析证实，蛋白质的“疏水区域”（第54 - 97位氨基酸残基，其中α-螺旋比例较大）对于二聚化至关重要，对于1 - 94位氨基酸残基的肽段，其表观解离常数K(D,app)约为50 μM，对于1 - 147位氨基酸残基的肽段，约为20 μM。我们的研究不排除可能形成少量额外的高阶复合物。
