蛋白质在执行其生物学功能时通常会形成多种构象态。蛋白质构象及变构动力学的探测和解析对于理解蛋白质结构与功能的关系至关重要。纳米孔技术是一种通过电流电阻测量来检测分子尺寸和形态变化的方法，具有水环境检测、高时空分辨率、无标记和高通量等优势。该技术已经在DNA测序、蛋白质识别和其他分子检测方面取得了重大突破。然而，在利用纳米孔技术来监测蛋白质结构时，还存在挑战。目前，蛋白质的构象一般是通过电流脉冲的幅度和时间宽度来反映，但是上述技术读出与特定构象态的结构并没有建立直观的对应关系，而且蛋白质过孔时的取向变化使得电流信号更加复杂，因此需要深入研究不同构象态和取向下的电流规律，以建立起电流变化与构象态之间的关联。另外，在构象涨落中蛋白质的取向也可能发生变化，中间态构象的结构与特征电流之间的对应关系尚不明确。因此仅根据电流脉冲的时空特征来推测中间态的结构仍然存在问题。此外，迫切需要研究纳米孔约束环境对蛋白质构象和变构过程的影响。基于上述问题，本工作选取αXβ2整合素为主要研究对象，采用分子动力学模拟（MD）为主要研究方法，旨在探讨纳米孔传感技术对αXβ2不同构象态和变构过程的探测和解析，主要涵盖以下两个方面： 1. 基于纳米孔阻塞电流解析蛋白质不同构象态的形貌特征标识。本工作基于全原子MD模拟建立了纳米孔检测典型整合素αXβ2不同构象态的模拟体系。首先通过对不同孔径、不同电场强度下纳米孔离子电流的分析，验证了模拟模型的正确性。其次，通过分析纳米孔相对阻塞电流，解耦了蛋白质构象和取向对纳米孔离子电流的调制，估计了αXβ2不同构象态结构的回转椭球体近似形貌。通过各构象态近似椭球体体积和形状的二维特征指标，成功区别了αXβ2的不同构象态。再次，分析了纳米孔中电导率的分布规律，探究了孔壁和蛋白质如何影响孔内电导率，并建立了改进的离散模型来精确计算纳米孔的电导。最后，将改进的离散模型与椭球近似相结合，提出了一种基于纳米孔传感的蛋白质形状估计方法。该方法从理论层面预测了αXβ2蛋白质的体积和形状，与MD模拟结果吻合。进一步，将该理论方法拓展至其他多种蛋白质的形貌估计，预测结果与文献报道的实验结果吻合较好，验证了该方法在纳米孔蛋白质检测中的适用性。综上，该工作能够通过蛋白质的阻塞电流，解析出分子结构的低分辨率椭球近似形貌，为纳米孔技术分析蛋白质结构提供了一种新方法。 2. 建立了将纳米孔传感与原子力显微镜（AFM）相结合的MD模拟体系，通过电学和力学耦合解析蛋白质变构的实时结构特征。首先，基于拉伸分子动力学（SMD）模拟，通过外力调控将纳米孔中的αXβ2由低亲和态拉伸为中亲和态。纳米孔中离子电流随变构过程逐渐变化，说明该模拟体系能够有效调控和准确表征蛋白质的变构。其次，基于纳米孔电流和AFM拉伸力二维信号，发展了一种实时估计变构过程中瞬时中间态近似椭球形貌的理论方法，并基于该理论方法，进一步模拟了αXβ2在不同直径的纳米孔中的力致变构动力学。结果表明，中间态构象的近似球状体的形状标识符足够敏感，可以区分从弯曲到直立状态的变构中，纳米孔不同约束条件下蛋白质的两种变构模式。最后，研究了纳米孔受限效应影响αXβ2变构的微观机理。纳米孔的强空间约束限制了构象伸展过程中关键结构域几何位置的变化，从而增大了从弯曲到直立状态所需克服的分子内相互作用的能垒。αXβ2颈-肩区域的伸长和股-腿关节的打开形成了对拉伸高度的竞争性补偿，两者之间的竞争会在不同直径的纳米孔和机械力拉伸速度下产生两种倾向性的变构模式。综上，该工作提出了一种实时可视化蛋白质变构动力学的新方法，能够描述中间态构象的结构特征，并评估纳米孔限域对蛋白质构象涨落的影响。 综上，本文探究了纳米孔离子电流与蛋白质构象之间的关联，并进一步建立了一种电学、力学结合的方法来解析蛋白质变构过程中瞬时中间态的结构。以上方法对纳米孔技术用于蛋白质构象和变构动力学的探测具有潜在的实用价值，对相应实验的设计与数据解析具有指导意义。本研究有利于促进纳米孔传感技术在蛋白质探测领域的应用。

    Proteins generally adopt various conformational states when carrying out their biological functions. The detection and analysis of protein conformation and conformational dynamics are crucial for understanding the relationship between protein structure and function. Nanopore technology is a method that utilizes current resistance measurements to detect changes in molecular size and morphology. It offers advantages such as aqueous environment detection, high spatiotemporal resolution, label-free detection, and high throughput, making significant breakthroughs in DNA sequencing, protein identification, and other molecular detection applications. However, challenges still remain when applying nanopore technology to monitor protein structures. Currently, the conformations of proteins are generally inferred from the amplitude and time width of current pulses. However, the correspondence between the readouts from these techniques and specific conformational states lacks a straightforward correlation. The orientation changes during protein translocation through the nanopore introduce the complexity to decipher the current signal. Thus, it is necessary to thoroughly study the current patterns under different conformational states and orientations and establish a connection between current fluctuations and conformational states. Furthermore, the orientation of proteins during the conformational fluctuation may also be changing, but the corresponding relationship between intermediate conformational states and characteristic current signatures remains unclear. Therefore, inferring the structure of intermediate states solely based on the spatiotemporal characteristics of current pulses still poses challenges. Additionally, investigating the influence of the nanopore-constrained environment on protein conformation and the allosteric process is urgently needed. In view of these issues, this work selects αXβ2 integrin as the primary research subject and employs molecular dynamics (MD) simulation as the main research method. The aim is to explore the detection and analysis of various conformational states and conformational dynamics of αXβ2 integrin using nanopore sensing technology. The main aspects of this work include the following two points:
    1. Resolution of morphological identifiers for distinct conformations via protein translocation current in nanopores. In this work, a nanopore sensing system is established for detecting distinct conformational states of the typical integrin protein αXβ2 using all-atom MD simulations. The validity of the simulation model is confirmed by analyzing nanopore ion currents under different pore sizes and electric field strengths. Subsequently, by analyzing the relative blockade currents, this work decouples the modulation of ionic currents in nanopores by protein conformations and orientations, and estimates the approximate spheroidal morphologies of αXβ2 conformational states. The distinct conformational states of αXβ2 are successfully differentiated by the two-dimensional characteristic identifiers of the approximated ellipsoidal volumes and shapes. Furthermore, this work analyzes the distribution patterns of conductivity within the nanopore, investigating how the pore wall and the protein itself affect the interior conductivity of the pore. Building upon this, an improved discrete model is established to accurately calculate the nanopore's conductivity. Lastly, by combining the improved discrete model with the spheroidal approximation, a nanopore-sensing-based protein conformation estimation method is proposed. This method theoretically predicts the volume and shape of the αXβ2 protein, and results are consistent with those MD outcomes. Additionally, this theoretical approach is extended to the morphological estimation of various other proteins, and the predicted results are in good agreement with the experimental results reported in literatures, verifying the applicability of this method in nanopore sening of proteins. Consequently, this work provides a means to deduce low-resolution spheroidal approximations of molecular structures through protein blockade currents, offering a new technique for protein structure analysis using nanopore.
    2. Real-time structural signatures of protein allosteric dynamics by electrically- and mechanically-coupled sensing. This work also establishes a MD simulation system that combines nanopore sensing technique with atomic force microscopy (AFM). Firstly, based on steered molecular dynamics (SMD) simulations, the external force is applied to stretch αXβ2 within the nanopore from a low-affinity state to an intermediate-affinity state.The nanopore ionic current gradually changes during the allosteric process, indicating the ability of this simulation system to accurately steer and detect protein conformational changes. Secondly, based on the two-dimensional signals of nanopore current and AFM tension force, this work develops a theoretical method for real-time estimation of the approximate spheroidal morphologies of transient intermediate states during the allosteric process. Based on this theoretical method, the stretched allostery of αXβ2 is simulated in nanopores of varying diameters. Results indicate that the shape identifiers of approximate spheroids for the intermediate state conformations are sufficiently sensitive to distinguish two allosteric patterns of proteins under different constraints of nanopores from bending to standing states. Finally, this work investigates the micro-mechanisms of nanopore-confined effects on the allosteric processes of αXβ2.The strong spatial constraint of the nanopore limits the geometric variations of key structural domains during the conformational expansion process, thereby increasing the energy barrier of intra-molecular interactions required to overcome for the transition from bending to standing state. The elongation of the neck-shoulder region and the opening of the thigh-calf joint result in a compensatory competition for the stretched height. This competition gives rise to alternative allosteric tendencies under different nanopore diameters and stretched velocities of mechanical probe. Therefore, this work proposes a new approach for real-time visualization of protein conformational dynamics during protein allostery, favoring to describe the structural signatures of intermediate state conformations and assess the impact of nanopore confinement on protein conformational fluctuation.
    In summary, this work investigates the relationship between nanopore ion currents and protein structures, and further establishes an integrated approach combining electrical and mechanical sensing to analyze intermediate structures during protein allostery. The results obtained potentiate significance for practical application of nanopore technology in detecting protein conformations and allosteric dynamics, and provide guidance for experimental design and data analysis. This work is beneficial for enhancing the application of nanopore sensing technology in the field of protein detection.