Membrane Protein Structure Elucidation from sparse NMR data (KAMP)

描述（由申请人提供）：虽然超过50%的药物靶向膜蛋白（MP），但蛋白质数据库（PDB）中保存的所有结构中只有不到1%属于这类蛋白质（仅约60个不同的折叠）。近年来，越来越多的膜蛋白结构已经通过先进的NMR光谱学得到解决。然而，稀疏的数据往往限制了这一有前途的途径结构解析。我们提出了一种新的基于计算知识的折叠组装算法的实现膜蛋白结构阐明“KAMP”。NMR数据的稀疏性将与从头蛋白质结构预测技术中基于知识的潜力相平衡，该技术通过跨膜区域中断开的二级结构元件的初始组装来规避早期方法的大小和复杂性限制。我们的方法将促进和实现更容易的NMR为基础的膜蛋白的结构阐明。这将促进对其功能的分子基础的理解。反过来，将促进药物和治疗方法的开发。E.大肠杆菌二酰基甘油激酶（DAGK）将被用作开发这种结合的计算/实验方法的主要车辆。目标一：开发KAMP用于从稀疏NMR限制中重新解析蛋白质折叠目的II：使用KAMP对已知结构的膜蛋白进行结构重建测定，用于方法验证和优化目的III：使用二酰基甘油激酶作为严格的测试用例来严格评估KAMP，并测量额外的实验限制对于该项目，我们可以立即获得大量的二酰基甘油激酶、人血型糖蛋白、外膜蛋白A和受磷蛋白五聚体。拟议研究的高计算和NMR光谱学需求将利用NIH对范德比尔特大学高级计算研究与教育中心（ACCRE）和生物分子NMR设施的投资。

DESCRIPTION (provided by applicant): While more than 50% of all drugs target membrane proteins (MPs), less than 1% of all structures deposited in the protein data bank (PDB) belong to this class of proteins (only about 60 distinct folds). An increasing number of membrane protein structures have been solved in recent years by advanced NMR spectroscopy. However, sparseness of data often limits this promising avenue of structure elucidation. We propose implementation of a novel computational knowledge-based fold assembly algorithm for membrane protein structure elucidation "KAMP". Sparseness of NMR data will be counter-balanced with knowledge-based potentials in a de novo protein structure prediction technique that circumvents the size and complexity limits of earlier methods via initial assembly of disconnected secondary structure elements in the transmembrane region. Our method will facilitate and enable more facile NMR-based structure elucidation of membrane proteins. This will promote understanding of the molecular basis for their function. In turn, development of drugs and therapeutics will be facilitated. E. coli Diacylglycerol kinase (DAGK) will be used as a primary vehicle for developing this combined computational/experimental approach. Aim I: Develop KAMP for de novo Protein Fold Elucidation from Sparse NMR Restraints Aim II: Reconstruct Structure Determination for Membrane Proteins of Known Structure with KAMP for Method Validation and Refinement Aim III: Critically Assess KAMP using Diacylglycerol Kinase as a Rigorous Test Case and Measure Additional Experimental Restraints For this project we have immediate access to considerable bodies of NMR data for diacylglycerol kinase, human glycophorin, outer membrane protein A, and the phospholamban pentamer through established collaborations. The high computational and NMR spectroscopy demands of the proposed research will leverage NIH investments in Vanderbilt University's Advanced Computing Center for Research & Education (ACCRE) and Biomolecular NMR Facility.