酰基辅酶A结合蛋白去折叠动力学的单分子磁镊研究

Single molecule magnetic tweezers for unfolding dynamics of Acyl-CoA binding protein

不同结构类型的蛋白质的力学稳定性和去折叠动力学有显著的差异,其中全部由α螺旋构成的蛋白质通常在较小的拉力作用下就会发生快速去折叠,需要能够精准控制皮牛量级拉力的实验手段来进行定量研究.酰基辅酶A结合蛋白(ACBP)是一种研究全α螺旋结构蛋白折叠/去折叠动力学性质的模型蛋白,其由86个氨基酸残基形成一种由4个α螺旋组成的螺旋束结构.本文利用磁镊对ACBP进行了恒定力加载速率的拉伸实验,得到了不同力加载速率下的去折叠力的分布.不同数据分析方法都显示ACBP具有超长的去折叠距离.对比分子动力学模拟拉伸的结果, ACBP的过渡态中整个N端α螺旋和部分的C端α螺旋发生了去折叠转变.本研究显示单分子拉伸实验与分子动力学模拟相结合是揭示蛋白质构象转变的分子机制的可靠研究方法.

There are significant differences in mechanical stability and unfolding dynamics among proteins with different structural compositions.Compared with proteins with β-sheets and subjected to shearing forces,proteins that are composed entirely of α-helices often undergo rapid unfolding under low stretching forces,thus requiring quantitative studies by using experimental tools that can precisely control forces on a pico-Newton scale.Magnetic tweezers with intrinsic force-control capability and great stability for long-time continuous measurement are suitable to measure force-induced conformation transitions of protein subjected to low forces of several pico-Newton.Acyl-CoA binding protein(ACBP)is a model protein used to study the folding/unfolding kinetics of complete α-helices protein.It is composed of 86 amino acid residues,forming a helical bundle of four α-helices.When its N-and C-terminal are stretched,the first and last a-helix are subjected to shear force in parallel.Previous biochemical studies showed that ACBP folding and unfolding in a two-state manner.In this paper,we use magnetic tweezers to stretch ACBP from its N-and C-end and obtain the distribution of the unfolding force at different loading rates ranging from 0.25 pN/s to 4 pN/s.The most probable unfolding forces are all less than 10 pN,which confirms that ACBP is not mechanically stable.At a constant loading rate,the unfolding force distribution and the most probable unfolding force as a function of loading rate have well-defined analytical formulas based on Bell’s model.Therefore,the experimental results of unfolding force can be fitted directly to obtain the important kinetic parameter of unfolding distance which is defined as the difference in extension between the native state and the transition state.Data analysis shows that ACBP has an extraordinarily long unfolding distance of 7.8 nm.Steered molecular dynamics simulations of ACBP stretching gives the transition state with N-terminal α-helix fully unfolded and C-terminal α-helix partially unfolded,which is consistent with the long unfolding distance obtained in the experiment on magnetic tweezers.According to the simulation results,the unfolding of α-helices is less cooperative than that of β-sheet structures.This characteristic makes α-helix proteins sensitive to mechanical forces,rendering them suitable as force sensors in cells.This study shows that single-molecule stretching experiment combined with molecular dynamics simulations is a reliable method to reveal the molecular mechanism of protein conformationtransitions under stretching forces.