酰基磷酸酶去折叠动力学的单分子磁镊研究
Single Molecule Magnetic Tweezers Study of Unfolding Kinetics of Acylphosphatase

DOI: 10.12677/BIPHY.2022.102003, PP. 22-30

Keywords: 磁镊，去折叠，过渡态，酰基磷酸酶
Magnetic Tweezers, Unfolding, Transition State, Acylphosphatase

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Abstract:

酰基磷酸酶(Acylphosphatase, AcP)是最小的基于蛋白质的生物酶之一，包含98个氨基酸残基，其天然构象由两个α螺旋和一个五链反平行的β折叠组成。AcP可以催化羧基–磷酸键的水解，在生物体代谢方面发挥着重要的生理作用。AcP是一种典型的两态折叠蛋白，它具有体积小、拓扑结构简单、分子内不存在二硫键的特点，是研究蛋白质折叠/去折叠机制的模型蛋白之一。在单分子力谱研究方面，人们已经用原子力显微镜研究了AcP的力学性能和去折叠行为。在本文中，基于稳定的磁镊单分子技术，我们测量了AcP在不同拉力下的去折叠速率。首先，我们构建了具有两种指纹信号(I27, SpyCatcher-SpyTag)的重组蛋白用于单分子操纵实验，然后通过拉力–伸长曲线中的特征确认正确的AcP去折叠信号，接下来开展了AcP在不同加载速率下的拉伸实验和恒定力下的拉伸实验。我们得到了AcP在20 pN到40 pN拉力区间内的去折叠速率，并分析了实验结果与原子力显微镜实验结果差异的原因与意义，结果暗示去折叠过渡态在不同拉力下会发生变化。
Acylphosphatase (AcP) containing 98 amino acid residues is one of the smallest globular enzymes. AcP can catalyze the hydrolysis of carboxyl-phosphate bonds and plays an important physiological role in the metabolic system of living organisms. The native state of AcP consists of two α helices and a five-stranded anti-parallel β sheet. AcP is a typical two-state protein, which has the characteristics of small size, simple topology and no disulfide bond in the molecule, which make it a model protein for studying the mechanism of protein folding/unfolding. The mechanical properties and unfolding behavior of AcP have been studied by atomic force microscopy (AFM). In this paper, based on the stable magnetic tweezers, we measured the unfolding rate of AcP at different constant stretching forces. Firstly, a recombinant protein construct with two kinds of fingerprint signals from I27 and SpyCatcher-SpyTag was produced for single-molecule manipulation experiment. The correct unfolding signal of AcP was identified through the fingerprint singles in the force-extension curve. Then we carried out stretching experiments of AcP at different loading rates and constant forces to obtain the unfolding rates of AcP from 20 pN to 40 pN. The difference between our results and the previous AFM experiment was discussed, and the result indicates that the unfolding transition state will change with force.

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