Incorporation of trifluoromethionine into a phage lysozyme: Implications and a new marker for use in protein F-19 NMR
Incorporation of trifluoromethionine into a phage lysozyme: Implications and a new marker for use in protein F-19 NMR
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DOI：
10.1021/bi9617973
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发表时间：
1997-03-18
期刊：
影响因子：
2.9
通讯作者：
Honek, JF
中科院分区：
文献类型：
--
作者：
Duewel, H;Daub, E;Honek, JF
目前人们的兴趣主要集中在了解特定氨基酸残基对蛋白质结构和功能的详细贡献上。虽然定点诱变的使用对这一目标做出了巨大贡献，但该方法仅限于20种氨基酸的标准曲目。氟化氨基酸已被成功地用于探测蛋白质的结构和动力学，并指出了特定残基对生物功能的重要性。在我们对氨基酸蛋氨酸在生物系统中的重要性的持续研究中，有报道成功地将L-S-（三氟甲基）同型半胱氨酸（l -三氟甲硫氨酸；L-TFM）掺入含有三个蛋氨酸残基的噬菌体lambda溶菌酶（LaL）中。以n -乙酰基- d、L-同型半胱氨酸硫内酯和碘化三氟甲基为原料，以33%的总收率合成了TFM的L异构体。制备了一种强过量表达LaL的表达质粒，并将其转化为蛋氨酸营养不良的大肠杆菌菌株，使其在L-TFM存在下表达LaL。该类似物不支持营养不良细胞的生长，并被发现对细胞生长有抑制作用。然而，最初在富met培养基中生长的细胞，在仔细控制培养基中L-Met和L-TFM的浓度下进行蛋白质诱导，能够在高（70%）和低（31%）TFM掺入水平下过表达TFM标记的LaL （TFM-LaL）。两种水平的TFM-LaL均表现出与野生型酶相似的活性，并被壳寡糖抑制，表明类似物的掺入不会阻碍酶的功能。有趣的是，tfm标记的酶的F-19溶液核磁共振光谱由四个跨越0.9 ppm化学位移的尖锐共振组成，其中三个共振显示出非常温和的壳五戊糖结合的屏蔽变化。在高和低掺入水平下对TFM-LaL的F-19核磁共振分析表明，其中一个蛋氨酸位置产生两个独立的共振。这两种共振的强度受到掺入程度的影响，这被解释为掺入TFM诱导蛋白质结构发生细微构象变化的迹象。利用从头算分子轨道计算分析了Met和TFM的异同。所提出的方法为研究蛋白质-配体相互作用以及未来研究蛋氨酸在蛋白质中的功能重要性提供了一种新的方法。
Much interest is currently focused on understanding the detailed contribution that particular amino acid residues make in protein structure and function. Although the use of site-directed mutagenesis has greatly contributed to this goal, the approach is limited to the standard repertoire of twenty amino acids. Fluorinated amino acids have been utilized successfully to probe protein structure and dynamics as well as point to the importance of specific residues to biological function. In our continuing investigations on the importance of the amino acid methionine in biological systems, the successful incorporation of L-S-(trifluoromethyl)homocysteine (L-trifluoromethionine; L-TFM) into bacteriophage lambda lysozyme (LaL), an enzyme containing three methionine residues, is reported. The L isomer of TFM was synthesized in an overall yield of 33% from N-acetyl-D,L-homocysteine thiolactone and trifluoromethyl iodide. An expression plasmid giving strong overproduction of LaL was prepared and transformed into Escherichia coli strain auxotrophic for methionine permitting the expression of LaL in the presence of L-TFM. The analogue would not support growth of the auxotroph and was found to be inhibitory to cell growth. However, cells that were initially grown in a Met-rich media followed by protein induction under careful control of the respective concentrations of L-Met and L-TFM in the media, were able to overexpress TFM-labeled LaL (TFM-LaL) at both high (70%) and low (31%) levels of TFM incorporation. TFM-LaL at both levels of incorporation exhibited analogous activity to the wild type enzyme and were inhibited by chitooligosaccharides indicating that incorporation of the analogue did not hinder enzyme function. Interestingly, the F-19 solution NMR spectra of the TFM-labeled enzymes consisted of four sharp resonances spanning a chemical shift ran of 0.9 ppm, with three of the resonances showing very modest shielding changes on binding of chitopentaose. The F-19 NMR analysis of TFM-LaL at both high and low levels of incorporation suggested that one of the methionine positions gives rise to two separate resonances. The intensities of these two resonances were influenced by the extent of incorporation which was interpreted as an indication that subtle conformational changes in protein structure are induced by incorporated TFM. The similarities and differences between Met and TFM were analyzed using ab initio molecular orbital calculations. The methodology presented offers promise as a new approach to the study of protein-ligand interactions as well as for future investigations into the functional importance of methionine in proteins.