Folding topology and DNA binding of the N‐terminal fragment of ada protein

FEBS Letters

Kainosho, Masatsune³  Sakuma, Takahiko³  Sakumi, Kunihiko¹  Sakashita, Hitoshi²  Morikawa, Kosuke²  Ohkubo, Tadayasu²  Sekiguchi, Mutsuo¹ 

[1] Medical Institute of Bio-Regulation, Kyushu University, Fukuoka 812, Japan;Protein Engineering Research Institute, 6-2-3, Furuedai, Suita, Osaka 565, Japan;Tokyo Metropolitan University, 1-1, Minami-ohsawa, Hachioji-shi, Tokyo, 192-03, Japan

关键词: Ada protein;    DNA binding activity;    Gel mobility shift assay;    Nuclear magnetic resonance;    Secondary structure;    N-ada 14k;    Ada fragment containing residues 1–129;    N-ada 16k;    Ada fragment containing residues 1–146;    N-ada 20k;    Ada fragment containing residues 1–178;    HMQC;    heteronuclear multi-quantum coherence;    PCR;    polymerase chain reaction;    SDS-PAGE;    sodium dodecyl sulfate polyacrylamide gel electrophoresis;    NOE;    nuclear Overhauser effect;   

DOI  :  10.1016/0014-5793(93)81351-Y

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Three amino terminal fragments of Escherichia coli Ada protein (39 kDa) with different molecular masses (14 kDa, 16 kDa and 20 kDa) were prepared in large quantities from an E. coli strain harboring plasmids constructed for the overproduction of the truncated proteins. The three fragments can be methylated to an extent similar to that of the intact molecule. The methylated 16 kDa fragment specifically binds to the ada box on a DNA duplex. NMR analyses revealed that the 14 kDa fragment comprises two α-helices and a β-sheet with parallel and anti-parallel mixed strands. A comparison of the ¹⁵N-¹H HMQC spectra of the fragments has led to the conclusion that this tertiary structure within the 14 kDa fragment is retained in the larger 16 kDa and 20 kDa fragments.

【 授权许可】

Unknown