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（MPs），但在蛋白质数据库（PDB）中沉积的所有结构中，只有不到1%属于这类蛋白质（只有大约60个不同的折叠）。近年来，越来越多的膜蛋白结构被先进的核磁共振光谱所解决。然而，数据的稀疏性往往限制了这种有前途的结构解释途径。我们提出了一种新的基于计算知识的折叠组装算法，用于膜蛋白结构解析“KAMP”。在一种新的蛋白质结构预测技术中，核磁共振数据的稀疏性将与基于知识的潜力相平衡，该技术通过在跨膜区域初始组装不连接的二级结构元素，绕过了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基于核磁共振的膜蛋白结构解析。这将促进对其功能的分子基础的理解。反过来，药物和治疗方法的开发将得到促进。大肠杆菌二酰基甘油激酶（DAGK）将被用作开发这种计算/实验结合方法的主要载体。目的一：开发KAMP用于稀疏核磁共振约束下的从头蛋白折叠解析；目的二：用KAMP重建已知结构膜蛋白的结构测定，用于方法验证和改进；目的三：使用二酰基甘油激酶作为严格的测试案例来严格评估KAMP，并测量额外的实验限制。在这个项目中，我们可以通过建立的合作关系，立即获得大量的二酰基甘油激酶、人糖蛋白、外膜蛋白a和磷五聚体的核磁共振数据。拟议研究的高计算和核磁共振光谱需求将利用美国国立卫生研究院对范德比尔特大学研究与教育高级计算中心（ACCRE）和生物分子核磁共振设施的投资。

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.