Computer-aided design of a catalyst for Edman degradation utilizing substrate-assisted catalysis
Computer-aided design of a catalyst for Edman degradation utilizing substrate-assisted catalysis
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DOI：
10.1002/pro.2633
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发表时间：
2014-12-16
期刊：
影响因子：
8
通讯作者：
Havranek, James J.
中科院分区：
文献类型：
--
作者：
Borgo, Benjamin;Havranek, James J.
以前对大样本进行的DNA测序分析的小型化和并行化使分子生物学发生了革命性的变化。其中许多技术依赖于生物分子试剂来促进样品的检测、合成或标记。为了帮助构建类似的蛋白质和多肽实验方法，我们使用计算机辅助设计来设计一种能够催化Edman降解的切割步骤的酶。我们利用Edman试剂上的硫亲核试剂和自然产生的蛋白酶中催化半胱氨酸的相似性，采用底物辅助的机制来实现N末端氨基酸的可控、逐步去除。在多肽的N-末端反复暴露氨基酸的能力是利用多肽链的过程降解的蛋白质测序技术的中心要求。虽然使用化学埃德曼降解可以很容易地实现这一点，但在水溶液中通过酶促实现这一活性无需苛刻的酸催化，从而改善了与低吸附检测表面的兼容性，例如用于单分子分析的表面。
Molecular biology has been revolutionized by the miniaturization and parallelization of DNA sequencing assays previously performed on bulk samples. Many of these technologies rely on biomolecular reagents to facilitate detection, synthesis, or labeling of samples. To aid in the construction of analogous experimental approaches for proteins and peptides, we have used computer-aided design to engineer an enzyme capable of catalyzing the cleavage step of the Edman degradation. We exploit the similarity between the sulfur nucleophile on the Edman reagent and the catalytic cysteine in a naturally occurring protease to adopt a substrate-assisted mechanism for achieving controlled, step-wise removal of N-terminal amino acids. The ability to expose amino acids iteratively at the N-terminus of peptides is a central requirement for protein sequencing techniques that utilize processive degradation of the peptide chain. While this can be easily accomplished using the chemical Edman degradation, achieving this activity enzymatically in aqueous solution removes the requirement for harsh acid catalysis, improving compatibility with low adsorption detection surfaces, such as those used in single molecule assays.
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