Factors which stabilize the methylamine dehydrogenase-amicyanin electron transfer protein complex revealed by site-directed mutagenesis
Factors which stabilize the methylamine dehydrogenase-amicyanin electron transfer protein complex revealed by site-directed mutagenesis
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DOI：
10.1021/bi971353m
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发表时间：
1997-10-21
期刊：
影响因子：
2.9
通讯作者：
Hosler, JP
中科院分区：
文献类型：
--
作者：
Davidson, VL;Jones, LH;Hosler, JP
甲胺脱氢酶 (MADH) 和阿米蓝蛋白形成生理复合物，其中电子从 MADH 的色氨酸色氨酸醌 (TTQ) 辅因子转移到阿米蓝蛋白的 1 型铜。负责复合物形成的相互作用可以从这些蛋白质复合物的晶体结构推断出来。已进行定点诱变来探测阿霉素的特定氨基酸残基在稳定 MADH-阿霉素复合物中的作用并确定观察到的复合物形成的离子强度依赖性。 Phe(97) 转化为 Glu 严重破坏了结合，证实了涉及该残基的疏水相互作用的重要性。在低离子强度下，Arg(99) 转化为 Asp 或 Leu 将复合物形成的 K-d 提高了 2 个数量级，从而确立了从涉及 Arg(99) 的晶体结构推断出的离子相互作用的重要性。 Lys(68) 向 Ala 的转化不会破坏低离子强度下的结合，但它确实大大减少了观察到的复合物形成的离子强度依赖性，这一点在野生型阿霉素中可见。这些结果表明，MADH 和阿霉素之间的生理相互作用通过离子和范德华相互作用的组合而稳定，并且蛋白质表面上的各个氨基酸残基能够决定这些可溶性氧化还原蛋白之间的特定相互作用。这些结果还表明，当MADH和阿霉素在溶液中彼此反应时，它们的取向与在结晶蛋白质复合物的结构中看到的蛋白质的取向相同。
Methylamine dehydrogenase (MADH) and amicyanin form a physiologic complex within which electrons are transferred from the tryptophan tryptophylquinone (TTQ) cofactor of MADH to the type 1 copper of amicyanin. Interactions responsible for complex formation may be inferred from the crystal structures of complexes of these proteins. Site-directed mutagenesis has been performed to probe the roles of specific amino acid residues of amicyanin in stabilizing the MADH-amicyanin complex and determining the observed ionic strength dependence of complex formation. Conversion of Phe(97) to Glu severely disrupted binding, establishing the importance of hydrophobic interactions involving this residue. Conversion of Arg(99) to either Asp or to Leu increased the K-d for complex formation by 2 orders of magnitude at low ionic strength, establishing the importance of ionic interactions which were inferred from the crystal structure involving Arg(99). Conversion of Lys(68) to Ala did not disrupt binding at low ionic strength, but it did greatly diminish the observed ionic strength dependence of complex formation that is seen with wild-type amicyanin. These results demonstrate that the physiologic interaction between MADH and amicyanin is stabilized by a combination of ionic and van der Waals interactions and that individual amino acid residues on the protein surface are able to dictate specific interactions between these soluble redox proteins. These results also indicate that the orientation of MADH and amicyanin when they react with each other in solution is the same as the orientation of the proteins which is seen in the structure of the crystallized protein complex.