Molecular Modeling of the Human P-glycoprotein Transporter Protein

与许多跨膜蛋白一样，通过X射线测定P-gp的结构 晶体学已经证明是非常困难的。这源于成型时遇到的问题 保持天然的物理化学环境， 蛋白质的不同部分，以及天然构象。经过多年的 通过奋进，最近一次获得了与小鼠P-gp蛋白密切相关的结构 年然而，许多问题仍然存在，如如何密切的晶体结构相关的 蛋白质在体内，以及构象如何变化作为运输功能的一部分。到 为了解决这些问题，我们正在努力整合所有可用的晶体学和 间接实验数据与物理化学为基础的数学方法，以产生 先进的结构模型。幸运的是，经过30多年的研究， 丰富的P-gp的信息，从中我们可以窥见P-gp的结构信息。此外 对于小鼠P-gp，晶体结构可从同源蛋白获得：特别是 细菌Sav 1866和MsbA脂质翻转酶。有用的间接实验数据示例 包括定点诱变的影响，天然存在的多态性， 残基交联。理论上的、基于物理化学的方法的例子包括 检查家族内残基保守性和极性/疏水性的模式 与MDR蛋白和ABC转运蛋白超家族密切相关。这些信息 有助于预测哪些残基暴露于膜的核心层和头基层， 哪些残基排列在孔中，哪些残基位于两个跨膜结构的界面处， 域.为此，我们正在开发同源家族的大序列比对， 超家族这一结果也将有助于确定 相关的突变，这有助于确定在基因组中邻近的残基组。 3-蛋白质的三维结构。最近，我们使用了我们的3D结构 人类P-gp的建模，以确定将电子顺磁性探针放置在何处 实验确定不同的构象状态的功能周期的 蛋白

Like many transmembrane proteins, determination of the structure of P-gp by X-ray crystallography has proven very difficult. This stems from the problems encountered forming sufficient-quality crystals that maintain the native physiochemical environments for the different parts of the protein, and thus the native conformations. After many years of endeavor, a structure of the closely-related mouse P-gp protein has become available this last year. However, many questions remain as to how close the crystal structure relates to the protein in vivo, and how the conformation changes as part of the transport function. To address these questions, we are striving to integrate all available crystallographic and indirect experimental data with physiochemically-based mathematical methods to produce advanced models of the structures. Fortunately, over three decades of study has provided a wealth of information about P-gp from which we can gleam structural information. In addition to mouse P-gp, crystallographic structures are available from homologous proteins: especially bacterial Sav1866 and the MsbA lipid flippase. Examples of useful indirect experimental data include the effects of site-directed mutagenesis, naturally occurring polymorphisms, and residue cross-linking. Examples of theoretical, physiochemically-based methods include examining the patterns of residue conservation and polarity/hydrophobicity within the family of closely related MDR proteins and the superfamily of ABC transporters. This information helps predict which residues are exposed to the core and headgroup layers of the membrane, which residues line the pore, and which are at the interfaces of the two transmembrane domains. To this end, we are developing a grand sequence alignment of homologous families and the superfamily. The results of this will also enable the determination of patterns of correlated mutations, which help identify groups of residues that are proximal in the 3-dimensional structure of the protein. Most recently we have used our 3-D structural modelling of human P-gp to determine where to put electron paramagentic probes to experimentally determine different conformational states over the functional cycle of the protein.