Membrane Protein Structure Elucidation from Limited Magnetic Resonance Data

摘要 大约1,110个非冗余序列家族，因此α- 螺旋完整膜蛋白(MPS)存在于序列数据库中。同时少于 蛋白质数据库(PDB)中已经储存了100个不同的MP褶皱。因此，对于大约1000α-螺旋MP 族至少有一个结构有待确定，以便明确指定一个折叠。而超过50%的 在所有靶向MPS的药物中，仍然很难获得高质量的MPS晶体，尽管已经有了 近年来，这一领域取得了令人瞩目的进展。即使MP可以结晶，但不是所有的官能态 可能被表示，集合状态被静态快照表示得很差，并且模型可能是 对结晶助剂感到不安。替代实验技术，如核磁共振和电子顺磁共振波谱 可能会对国会议员造成结构性限制。但是，数据集通常保持稀疏，并且与 误差率。数据的稀疏性导致对计算方法进行集成的需求增加 稀疏来自多种技术的数据，并补充缺失的实验信息。 在这里，我们提出了一种集成在生化反应中的MP结构确定算法的开发 软件库(BCL)“BCL：：MP-Fold”具有以下亮点：a)实验数据的稀疏性是 通过将多种实验方法与基于知识的潜力相结合来实现平衡。B) 通过从头折叠算法解决了许多MPS缺乏合适的模板的问题。C)大小和 当前计算方法的复杂性限制是通过对断开连接的秒序列的初始组装来绕过的。 跨膜区的二级结构元素(SSE)。D)计算工作得到补充 通过包括非天然氨基酸的顺磁标记策略来产生核磁共振和EPR限制， 克服半胱氨酸标记策略的局限性，避免繁琐且容易出错的 核磁共振信号传递给侧链原子，特别适合用于bCL：：MP-折叠。最终， BCL：：MP-折叠将有助于确定质子化的α-螺旋MP的结构 每个Promier有四个跨膜跨度。埃姆雷质子态是一种重要的介质态。 在小的多药转运体的运输周期中的状态，尚待确定的三个- 空间结构。

SUMMARY Approximately 1110 non-redundant sequence families and therefore potentially distinct folds for α- helical integral membrane proteins (MPs) are represented in sequence databases. At the same time less than 100 distinct MP folds have been deposited in the protein data bank (PDB). Thus, for around 1000 α-helical MP families at least one structure remains to be determined to unambiguously assign a fold. While more than 50% of all drugs target MPs it remains difficult to obtain high-quality crystals of MPs, even though there has been spectacular progress in this area in recent years. Even if a MP can be crystallized, not all functional states might be represented, ensemble states are badly represented by static snapshots, and the model might be perturbed by crystallization aides. Alternative experimental techniques such as NMR and EPR spectroscopy can result in structural restraints for MPs. However, datasets typically remain sparse and are affiliated with an error margin. This sparseness of data leads to an increased demand for computational methods to integrate sparse data from multiple technologies and supplement missing experimental information. Here we propose development of a MP structure determination algorithm integrated in the BioChemical software Library (BCL) “BCL::MP-Fold” with the following highlights: a) Sparseness of experimental data is counter-balanced by integrating multiple experimental approaches with knowledge-based potentials. b) The absence of suitable templates for many MPs is addressed through a de novo folding algorithm. c) The size and complexity limits of current computational methods are circumvented via initial assembly of disconnected sec- ondary structure elements (SSEs) in the trans-membrane region. d) The computational effort is complemented by a paramagnetic tagging strategy including non-natural amino acids to yield NMR and EPR restraints, that overcome limitations of cysteine labeling strategies, circumvent the tedious and error-prone assignment of NMR signals to side chain atoms, and are especially well suited for use in BCL::MP-Fold. Ultimately, BCL::MP-FOLD will help to determine the structure of protonated EmrE, a homo-dimeric α-helical MP with four trans-membrane spans per protamer. The protonated state of EmrE is an important interme- diate in the transport cycle of the small multidrug transporter with a yet to be determine three- dimensional structure.