Site-specific incorporation of fluorescent probes into protein:: Hexahistidine-tag-mediated fluorescent labeling with (Ni2+:Nitrilotriacetic acid)n-fluorochrome conjugates
Site-specific incorporation of fluorescent probes into protein:: Hexahistidine-tag-mediated fluorescent labeling with (Ni2+:Nitrilotriacetic acid)n-fluorochrome conjugates
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DOI：
10.1021/ja017074a
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发表时间：
2001-12-05
影响因子：
15
通讯作者：
Ebright, RH
中科院分区：
文献类型：
--
作者：
Kapanidis, AN;Ebright, YW;Ebright, RH
通过荧光共振能量转移（FRET）1，2的蛋白质的结构和机械表征需要在特定的，确定的位点掺入荧光探针的能力。2对于不含半胱氨酸残基的蛋白质，位点特异性荧光标记可以通过使用定点诱变在感兴趣的位点引入半胱氨酸残基，然后通过半胱氨酸特异性化学修饰掺入荧光探针来实现。2然而，对于含有半胱氨酸残基的蛋白质（大多数MW> 50 kDa的蛋白质），位点特异性荧光标记是困难的。已经报道了三种策略：（i）内含肽介导的标记（"表达蛋白连接"），3（ii）氧化介导的标记，4和（iii）三价砷介导的标记。前两种策略仅限于标记蛋白质末端，不允许原位标记（即，直接标记比色皿、凝胶、印迹或生物样品中的蛋白质，而不需要随后的纯化步骤）;第三种策略目前仅限于单一荧光染料。在这里，我们报告了一种策略，允许标记的终端或内部网站，允许原位标记，并与一系列具有不同的光谱和物理性质的荧光染料兼容。我们的策略涉及使用"六组氨酸标签" 6、7、8 s即氨基酸序列His6s来靶向位点特异性荧光标记。已知六组氨酸标签与过渡金属络合物紧密相互作用，包括Ni 2+：次氮基三乙酸（Ni 2+：NTA）。6-8六聚组氨酸标签可以通过使用标准分子生物学程序6引入到蛋白质末端或内部位点，并广泛用于分子生物学研究中，用于基于亲和层析的蛋白质纯化[使用（Ni 2+：NTA）-琼脂糖] 6和蛋白质固定化[使用（Ni 2+：NTA）涂层表面]。6，7我们假设六聚组氨酸标签应该与（Ni2+：NTA）n-荧光染料结合物紧密相互作用，因此应该能够介导位点特异性荧光标记（图1）。我们进一步假设，基于分子建模，六聚组氨酸标签应该能够与多达两个Ni 2+：NTA部分相互作用，而没有空间位阻。为了检验这些假设，我们制备并分析了（Ni2+：NTA）1-荧光染料缀合物和（Ni2+：NTA）2-荧光染料缀合物。我们合成了具有一个侧基Ni~（2+）：NTA-结构的广泛使用的菁荧光染料Cy_3和Cy_52，9的衍生物。（Ni2+：NTA）1-Cy3和（Ni2+：NTA）1-Cy5;图1a中的1a和1b]或两个侧基Ni2+：NTA-部分[（Ni2+：NTA）2-Cy3和（Ni 2+：NTA）2-Cy5;通过Cy3和Cy59的单-和双琥珀酰亚胺基-酯衍生物与N-（5-氨基-1-羧基戊基）亚氨基二乙酸，10，然后与NiCl 2反应（图1a，B;表1）。
Structural and mechanistic characterization of proteins by fluorescence resonance energy transfer (FRET) 1, 2 requires the ability to incorporate fluorescent probes at specific, defined sites. 2 For proteins that do not contain cysteine residues, site-specific fluorescent labeling can be accomplished by use of site-directed mutagenesis to introduce a cysteine residue at the site of interest, followed by cysteine-specific chemical modification to incorporate the fluorescent probe. 2 However, for proteins that contain cysteine residues (most proteins with MW> 50 kDa), site-specific fluorescent labeling is difficult. Three strategies have been reported:(i) intein-mediated labeling (“expressed protein ligation”), 3 (ii) oxidation-mediated labeling, 4 and (iii) trivalent-arsenicmediated labeling. 5 The first two strategies are limited to labeling of protein termini and do not permit in situ labeling (ie, direct labeling of proteins in cuvettes, gels, blots, or biological samplesswithout the need for a subsequent purification step); the third strategy currently is limited to a single fluorochrome. Here, we report a strategy that permits labeling of termini or internal sites, that permits in situ labeling, and that is compatible with a range of fluorochromes with different spectroscopic and photophysical properties. Our strategy involves use of the “hexahistidine tag” 6, 7, 8 s ie, the amino acid sequence His6s to target site-specific fluorescent labeling. The hexahistidine tag is known to interact tightly with transition-metal complexes, including Ni2+: nitrilotriacetic acid (Ni2+: NTA). 6-8 The hexahistidine tag can be introduced at protein termini or internal sites by using standard molecular-biology procedures6 and is widely used in molecular-biology research for affinity-chromatography-based protein purification [with (Ni2+: NTA)-agarose] 6 and protein immobilization [with (Ni2+: NTA)-coated surfaces]. 6, 7 We hypothesized that the hexahistidine tag should interact tightly with (Ni2+: NTA) n-fluorochrome conjugates and thus should be able to mediate site-specific fluorescent labeling (Figure 1). We further hypothesized, based on molecular modeling, that the hexahistidine tag should be able to interact with up to two Ni2+: NTA moieties without steric hindrance. To test these hypotheses, we prepared and analyzed (Ni2+: NTA) 1-fluorochrome conjugates and (Ni2+: NTA) 2-fluorochrome conjugates. We synthesized derivatives of the widely used cyanine fluorochromes Cy3 and Cy52, 9 having one pendant Ni2+: NTA-moiety [(Ni2+: NTA) 1-Cy3 and (Ni2+: NTA) 1-Cy5; 1a and 1b in Figure 1a] or two pendant Ni2+: NTA-moieties [(Ni2+: NTA) 2-Cy3 and (Ni2+: NTA) 2-Cy5; 2a and 2b in Figure 1b] by reaction of mono-and bissuccinimidyl-ester derivatives of Cy3 and Cy59 with N-(5-amino-1-carboxypentyl) iminodiacetic acid, 10 followed by reaction with NiCl2 (Figure 1a, b; Table 1).