Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Republic of Korea.
Department of Biochemistry, College of Natural Sciences, Chungnam National University, Daejeon 34134, Republic of Korea.
ACS Sens. 2022 Jan 28;7(1):131-141. doi: 10.1021/acssensors.1c01819. Epub 2021 Dec 22.
Methionine oxidation is involved in regulating the protein activity and often leads to protein malfunction. However, tools for quantitative analyses of protein-specific methionine oxidation are currently unavailable. In this work, we developed a biological sensor that quantifies oxidized methionine in the form of methionine--sulfoxide in target proteins. The biosensor "tpMetROG" consists of methionine sulfoxide reductase B (MsrB), circularly permuted yellow fluorescent protein (cpYFP), thioredoxin, and protein G. Protein G binds to the constant region of antibodies against target proteins, specifically capturing them. Then, MsrB reduces the oxidized methionine in these proteins, leading to cpYFP fluorescence changes. We assessed this biosensor for quantitative analysis of methionine--sulfoxide in various proteins, such as calmodulin, IDLO, LegP, Sacde, and actin. We further developed an immunosorbent assay using the biosensor to quantify methionine oxidation in specific proteins such as calmodulin in animal tissues. The biosensor-linked immunosorbent assay proves to be an indispensable tool for detecting methionine oxidation in a protein-specific manner. This is a versatile tool for studying the redox biology of methionine oxidation in proteins.
蛋氨酸氧化参与调节蛋白质活性，通常会导致蛋白质功能障碍。然而，目前还没有用于定量分析蛋白质特异性蛋氨酸氧化的工具。在这项工作中，我们开发了一种生物传感器，可定量分析目标蛋白中蛋氨酸-亚砜形式的氧化蛋氨酸。该生物传感器“tpMetROG”由蛋氨酸亚砜还原酶 B（MsrB）、环化黄色荧光蛋白（cpYFP）、硫氧还蛋白和蛋白 G 组成。蛋白 G 结合到针对目标蛋白的抗体的恒定区，特异性地捕获它们。然后，MsrB 将这些蛋白质中的氧化蛋氨酸还原，导致 cpYFP 荧光变化。我们评估了该生物传感器对各种蛋白质（如钙调蛋白、IDLO、LegP、Sacde 和肌动蛋白）中蛋氨酸-亚砜的定量分析。我们进一步开发了一种使用该生物传感器的免疫吸附测定法，以定量测定动物组织中特定蛋白质（如钙调蛋白）中的蛋氨酸氧化。生物传感器连接的酶联免疫吸附测定法证明是一种不可或缺的工具，可特异性检测蛋白质中的蛋氨酸氧化。这是一种用于研究蛋白质中蛋氨酸氧化的氧化还原生物学的多功能工具。
