Peptidyl linkers for protein heterodimerization catalyzed by microbial transglutaminase.
Peptidyl linkers for protein heterodimerization catalyzed by microbial transglutaminase.
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微生物转谷氨酰胺酶催化蛋白质异二聚化的肽基接头。
DOI：
10.1021/bc034209o
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发表时间：
2004
影响因子：
4.7
通讯作者：
Teruyuki Nagamune
中科院分区：
文献类型：
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作者：
Tsutomu Tanaka;N. Kamiya;Teruyuki Nagamune
基于核糖核酸酶S-肽，使用定点诱变，产生导致功能蛋白质异源二聚化的特异性肽基接头，所述异源二聚化由来自茂原链霉菌（Streptomyces mobaraensis，MTG）的微生物转氨酶催化。肽基接头被指定为Lys-标签和Gln-标签，其被设计为具有唯一的反应性Lys或Gln残基，其适合于不同蛋白质的选择性Lys-Gln交联。采用绿色荧光蛋白变体ECFP和EYFP作为模型蛋白，并将这些Lys-和Gln-标签分别融合到ECFP和EYFP的N-末端。结果，我们成功地单独获得了ECFP-EYFP异二聚体，而没有形成多重交联的副产物。发现肽基接头的反应性根据待替换的氨基酸的类型而变化。具有碱性氨基酸（Arg）的肽基接头在交联反应中表现出最高的反应性，表明MTG的阳离子残基底物偏好。利用荧光共振能量转移（FRET）的动力学分析（其仅在异二聚体ECFP-EYFP缀合后观察到）揭示了氨基酸置换通过增加催化转换（k（cat））而不是底物结合亲和力（K（m））来促进交联反应的加速。最后，使用核糖核酸酶S-蛋白质，基于特定的S-肽：S-蛋白质相互作用的酶促蛋白质交联的操作进行了探索。由于新设计的Lys-和Gln-标签保留了对S-蛋白的结合亲和力，异源二聚化通过用S-蛋白包裹它们而被完全抑制。结果表明，有限的蛋白质结合的可能性，通过调整空间位阻对MTG。无论是工程肽基底物或通过考虑特定的肽-蛋白质相互作用，定制酶的翻译后修饰可能有助于开发一种新的顺序蛋白质缀合方法用于制备多功能蛋白质。
Specific peptidyl linkers that result in the heterodimerization of functional proteins, which is catalyzed by microbial transglutaminase from Streptomyces mobaraensis (MTG), were generated based on a ribonuclease S-peptide using site-directed mutagenesis. The peptidyl linkers designated as Lys-tag and Gln-tag were designed to possess sole reactive Lys or Gln residue that was amenable for selective Lys-Gln cross-linkage of different proteins. Green fluorescent protein variants, ECFP and EYFP, were employed as model proteins, and those Lys- and Gln-tags were fused to the N-termini of ECFP and EYFP, respectively. As a result, we succeeded in solely obtaining the ECFP-EYFP heterodimer without forming multiply cross-linked byproducts. It was found that the reactivity of peptidyl linkers varied according to the type of amino acid to be replaced. Peptidyl linkers with a basic amino acid (Arg) exhibited the highest reactivity in the cross-linking reaction, suggesting the cationic residue substrate preference of MTG. Kinetic analysis utilizing fluorescent resonance energy transfer (FRET), that is only observed upon the heterodimeric ECFP-EYFP conjugation, revealed that the amino acid replacement contributed to the acceleration of cross-linking reactions by increasing catalytic turnover (k(cat)), rather than substrate binding affinity (K(m)). Finally, using a ribonuclease S-protein, the manipulation of enzymatic protein cross-linking based on specific S-peptide:S-protein interactions was explored. Since newly designed Lys- and Gln-tags retained binding affinities to the S-protein, the heterodimerization was perfectly restrained by wrapping them with the S-protein. The results suggest the possibility of limited protein conjugation by tuning steric hindrance against the MTG. Tailoring enzymatic posttranslational modifications with either engineering peptidyl substrates or by taking specific peptide-protein interactions into consideration may facilitate the development of a new sequential protein conjugation method for the preparation of multifunctional protein.