Search for the Structural Basis of Biomacromolecular Function and Activity

(1)。我们在RAP中发现了一种新的组氨酸开关，细胞利用它在受体成熟过程中介导低密度脂蛋白受体相关蛋白(LRP)的结合/释放。细胞，2006，22(3)：423-30)。组氨酸开关TRAIL的发现开创了一种隔离受体的新方法。我们在研究过程中制作的突变体在治疗与LRP受体相关的疾病方面具有潜在的治疗应用。在这个项目的过程中，我们还完成了RAP蛋白的结构测定。RAP是一种蛋白质伴侣，在细胞内低密度脂蛋白受体的折叠/运输中发挥重要作用。由于RAP结构的性质，确定RAP的3D结构一直是极其困难的。我们的团队已经成功地使用溶液核磁共振和小角中子散射(SANS)相结合的方法确定了RAP的3D结构。RAP的3D结构提供了对蛋白质多功能性的结构基础的第一次洞察。这一结果已发表在PROT上。科学，2007，16(8)：1628-40。我们将继续研究LRP受体在Wnt/β-catenin信号通路中的作用。我们利用昆虫细胞成功地克隆并表达了LRP5和LRP6(LRP5/6)受体的可溶胞外区。目前，我们正在研究一种实用的程序，以大规模纯化受体。我应该指出，我们在这方面的研究步伐取决于分配给我所在部门的资源，而且受到资源的限制。(2)。我们完成了蛋白质L11在游离态、L11-RNA二元体和L11-RNA-硫代链球菌三元复合体中的结构和动力学研究，揭示了L11在蛋白质合成中调控伸长过程中各组分运动的结构和动力学基础。这一结果对于理解蛋白质合成中伸长/易位反应的机制具有重要意义。这项研究已经发表在J.Mol上。比奥尔。2007年、367(4)、1007-1022。(3)。我们已经完成了3P程序的开发。3P程序使用来自单个排列张量的剩余偶极耦合来确定蛋白质的主干结构。一个J.Magn。文中详细介绍了3P法的理论处理方法和程序。(4)。3UTRRNA(100个核苷酸)的结构测定正在顺利进行。目前，我们已经用各种同位素标记和突变方案制作了几个核磁共振样品；我们在国家Argonne实验室收集了几个RNA结构的高质量X射线小角和广角散射数据集；我们正在开发在散射数据限制下精炼RNA结构所需的计算协议；我们正在重新编码一组溶液核磁共振谱，这是阐明溶液中RNA分子结构细节所必需的。我们已经开发了3UTRRNA的初始结构模型。

(1). We have discovery a novel histidine switch in RAP that cell uses to mediate the binding/release of the low-density lipoprotein receptor related protein (LRP) in the receptor maturation process (Mol. Cell, 2006, 22(3):423-30). The discovery the histidine switch trail blazes a new way to sequester receptors. The mutants that we have made during the course of the study have potential therapeutic applications in treatment of diseases involved with the LRP receptors. During the course of this project, we have also completed the determination of the structure of protein RAP. RAP is a protein chaperone and plays an important role in folding/trafficking of the low-density lipoprotein receptors in cells. Due to the nature of the RAP structure, it has been extremely difficult to determine the 3D structure of RAP. Our group has successfully determined the 3D structure of RAP using a combination of the solution NMR and the Small Angle Neutron Scattering (SANS). The 3D structure of RAP provides the first insight into the structural basis of versatility of the protein. This result has been published in Prot. Sci., 2007, 16(8):1628-40. We will continue our studies of the roles of the LRP receptors in the Wnt/beta-catenin signaling pathway. We have successfully cloned and expressed the soluble extracellular domains of the LRP5 and LRP6 (LRP5/6) receptors on a large scale using the insect cells. At the present time, we are working on a practical procedure to purify the receptors on a large scale. I should point out that the pace of our research in this area depends on and is limited by the resource that was allocated to my section. (2). We have completed the structural and dynamics studies of protein L11 in free, in the L11-RNA binary and the L11-RNA-thiostrepton ternary complexes, and revealed the structural and dynamics basis for L11 to gate the movements of various components involved in the elongation in protein synthesis. This result is of fundamental importance in understanding the mechanism of the elongation/translocation reaction in the protein synthesis. This work has been published in J. Mol. Biol. 2007, 367(4), 1007-1022. (3). We have completed the development of the 3P program. The 3P program uses the residual dipolar couplings from a single alignment tensor to determine the backbone structure of a protein. A J. Magn. Res. article that presents a detailed description of rigorous theoretical treatment and the programs of the 3P method is in press. (4). The determination of the structure of a 3UTR RNA (100 nt) is well underway. At the present time, we have made several NMR samples with various isotope-labeling and mutation schemes; we have collected high quality X-ray small and wide angle scattering data sets of several RNA constructs at the National Argonne Laboratory; we are developing the computational protocol that is required to refine RNA structures with restraints of scattering data; we are recoding an arrays of solution NMR spectra that are required to elucidate structural details of RNA molecules in solution.; we have developed the initial structural model of the 3UTR RNA.