Firefly luciferase: Alteration of the color of emitted light resulting from substitutions at position 286
Firefly luciferase: Alteration of the color of emitted light resulting from substitutions at position 286
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DOI：
10.1021/ja961053c
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发表时间：
1996-07-31
影响因子：
15
通讯作者：
Hecht, SM
中科院分区：
文献类型：
--
作者：
Mamaev, SV;Laikhter, AL;Hecht, SM
萤火虫荧光素酶转换化学能以产生光。[1]有趣的是，不同种类的萤火虫，甚至同一只萤火虫的不同解剖部位，都能发出不同颜色的光，尽管它们的发光酶都使用相同的底物。[2]光的产生涉及到萤火虫荧光素向萤光素的净转化;激发态的萤光素（二）阴离子被认为实际上发出了观察到的光。[3]几条不同的证据表明，发射光的波长广泛地受结合于ATP的蛋白质的结构和构象的控制，包括在存在“伸展”ATP类似物的情况下发射光的颜色的pH依赖性改变3b，以及没有任何共价酶-底物中间体的证据。Kajiyama和中野确定了十字Luciola萤光素酶中的五个位点，在这些位点处，单个氨基酸的变化可以显著改变发射光的波长。[4]这五个位点在整个蛋白质序列中被广泛分离，这进一步支持了蛋白质结构的许多改变可以影响发射波长的观点。荧光素酶点突变体发射比野生型更长波长的趋势与破坏底物-荧光素酶相互作用的非特异性改变一致，例如通过提高系统的基态能量。为了进一步确定发光的性质，我们研究了Luciola mingrelica荧光素酶，一种不耐热的物种。5，6使用几种错酰化的抑制性tRNA 8抑制在位置286处引入的TAG密码子7，从而提供其中Ser 286被其它氨基酸取代的蛋白质。目前，我们证明了（i）第一次糖基化和磷酸化的氨基酸直接掺入到新生酶，（ii）在L。mingrelia荧光素酶突变体的发光效率可以是温度依赖性的，并且受Ser 286改变的影响不同。将mingrelica，5，9克隆到表达载体pTrc-99 A10中，在trc启动子的控制下。使用位点特异性诱变将第286位的Ser密码子AGT改变为终止密码子TAG。最初使用大肠杆菌菌株测定286位丝氨酸的其他天然氨基酸的取代，每种菌株含有由不同内源性氨酰-tRNA合成酶识别的特异性抑制tRNA。11.用质粒pTrcLuc-St 286转化这些菌株后，测量由每种精心加工的内切酶发射的光谱。12，13野生型荧光素酶和E.含有抑制tRNASer的大肠杆菌菌株产生在约583 nm处具有最大发射的光。其他10个氨基酸取代中的每一个都提供了荧光素酶，其发射的光显示出改变的λmax;表1中给出了几个代表性的例子。如图所示，所有修饰的双链酶发射的光的波长都比野生型长。最大的差异是对于含有Ser 286（582 nm）和Leu 286（621 nm）的酶（图1）;这对应于大约3 kcal/mol的能量差。
Firefly luciferase transduces chemical energy for the production of light. 1 Interestingly, different species of fireflies, and even different anatomical regions of a single firefly, can emit light of different colors, athough their luciferases all use the same substrate. 2 Light production involves the net conversion of firefly luciferin to oxyluciferin; an excited state oxyluciferin (di) anion is believed to actually emit the observed light. 3 That the wavelength of emitted light is controlled broadly by the structure and conformation of the protein bound to luciferin is suggested by a few different lines of evidence, including altered pH dependence of the color of emitted light in the presence of a “stretched-out” ATP analogue3b and the absence of evidence for any covalent enzyme-substrate intermediate. Kajiyama and Nakano identified five sites in Luciola cruciata luciferase at which a single amino acid change could substantially alter the wavelength of emitted light. 4 That these five sites were widely separated throughout the sequence of the protein further supports the idea that numerous alterations of protein structure can influence the emission wavelength. The tendency for luciferase point mutants to emit longer wavelengths than the wild type is consistent with nonspecific alterations that destabilize the substrate-luciferase interaction, eg by raising the ground state energy of the system. To further define the nature of light emission, we have studied Luciola mingrelica luciferase, a thermolabile species. 5, 6 Suppression of a TAG codon7 introduced at position 286 was effected using several misacylated suppressor tRNAs, 8 affording proteins in which Ser286 was replaced with other amino acids. Presently we demonstrate (i) the first direct incorporation of glycosylated and phosphorylated amino acids into a nascent enzyme,(ii) that replacement of Ser286 in L. mingrelia luciferase can result in alteration of the wavelength of emitted light, and (iii) that the efficiency of light emission by the derived luciferase mutants can be temperature dependent and affected differentially by alteration of Ser286.The luciferase gene of L. mingrelica, 5, 9 was cloned into expression vector pTrc-99A10 under the control of a trc promoter. Site-specific mutagenesis was used to change Ser codon AGT at position 286 to stop codon TAG. Substitution of other naturally occurring amino acids for serine at position 286 was assayed initially using Escherichia coli strains, each of which contained a specific suppressor tRNA recognized by a different endogenous aminoacyl-tRNA synthetase. 11 Following transformation of these strains with plasmid pTrcLuc-St286, the spectrum of light emitted by each of the elaborated luciferases was measured. 12, 13 Both wild-type luciferase and luciferase elaborated by the E. coli strain containing suppressor tRNASer produced light with an emission maximum at about 583 nm. Each of 10 other amino acid substitutions afforded a luciferase whose emitted light exhibited an altered λmax; several representative examples are given in Table 1. As shown, all of the modified luciferases emitted light at longer wavelengths than wild type. The greatest difference was for the luciferases containing Ser286 (582 nm) and Leu286 (621 nm)(Figure 1); this corresponds to an energy difference of approximately 3 kcal/mol.