ATP结合模块（ATP-binding cassette,ABC）转运体家族蛋白负责主动输运物质进出细胞膜，参与细胞的诸多重要生理过程，也是临床治疗中细菌和癌细胞抗药性的主要来源。维生素B12转运体蛋白BtuCD是ABC转运体家族转运机理研究的典型体系，本项目计划运用分子动力学模拟方法在原子水平上研究BtuCD膜蛋白构象变化的热力学和动力学，阐明底物转运机制。主要采用metadynamics等增强抽样方法计算BtuCD四个不同构象之间沿特定途径转换的自由能变化曲线（面），得到转运循环中构象变化的完整图像。研究底物结合蛋白BtuF和核苷酸分子对BtuCD构象变化的影响，澄清二者对BtuCD转运循环的调控作用。通过计算转运循环中关键蛋白构象和底物结合的自由能，阐明构象变化和底物转运的关系。分析底物转运机制中的关键氨基酸残基，通过设计突变进行实验验证，深入揭示底物转运的分子机制。

ATP-binding cassette(ABC)transporters actively translocate substances across cell membranes, involving in many important biological processes.They are also implicated in multidrug resistences in bacteria infection and cancer therapy. Vitamin B12 transporter BtuCD is a typical model system in the study of molecular mechanism of ABC transporters. This project aims to study the thermodynamics and kinetics of conformational movement of BtuCD at atomic level using molecular simulation methods. We will calculate the free energy profiles of conformational transition among the four conformations of BtuCD using enhanced sampling methods such as adaptive biasing force and metadynamics, obtaining the overall picture of conformational movement during translocation cycle of BtuCD. In order to elucidate the regulation roles of BtuF and nucleotides to BtuCD conformational transition, we will calculate the free energy change upon their association to the transporter. We will calculate the binding energy of substrate to the key conformations suring translocation cycle to reveal the relationship between protein conformational change and substrate translocation. By identifying the critical residues for substrate translocation, mutagenesis experiments and functional asssays will be performed to elicidate the translocation mechanism thoroughly.