Optimization of amino acid type-specific 13C and 15N labeling for the backbone assignment of membrane proteins by solution- and solid-state NMR with the UPLABEL algorithm
Optimization of amino acid type-specific 13C and 15N labeling for the backbone assignment of membrane proteins by solution- and solid-state NMR with the UPLABEL algorithm
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DOI：
10.1007/s10858-010-9462-4
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发表时间：
2011-02-01
影响因子：
2.7
通讯作者：
Guentert, Peter
中科院分区：
文献类型：
--
作者：
Hefke, Frederik;Bagaria, Anurag;Guentert, Peter
我们提出了一种计算方法，为膜蛋白和其他传统策略无法分配的大型蛋白质的骨架分配找到最优标记模式。遵循Kainosho和Tsuji的方法(生物化学21：6273-6279(1982))，不同类型的氨基酸用C-13或/和N-15标记，使得(CO)-C-13(I-1)和(NH)-N-15(I)之间的交叉峰只产生顺序相邻的氨基酸对，其中第一个用C-13标记，第二个用N-15标记。通过这种方式，可以对蛋白质序列中恰好出现一次的独特的氨基酸对进行明确的序列特异性分配。实际上，在获得标记唯一氨基酸对的最佳范围的同时，限制必须制备的不同标记蛋白质样本的数量是至关重要的。我们的用于最佳唯一对标记的计算机算法UPLABEL在程序CYANA和独立程序中实现，也可通过门户网站获得，它使用组合优化来寻找给定氨基酸序列的标记模式，该模式使用最少数量的不同标记蛋白质样本来最大化唯一对分配的数量。在确定最佳的氨基酸类型特定标记模式时，可以考虑各种辅助条件，包括标记氨基酸的可用性和价格、先前已知的部分分配和特别感兴趣的序列区域。用该方法对人G蛋白偶联受体缓激肽B2(B2R)进行了归属，并作为膜蛋白视紫红质骨架归属的起点。
We present a computational method for finding optimal labeling patterns for the backbone assignment of membrane proteins and other large proteins that cannot be assigned by conventional strategies. Following the approach of Kainosho and Tsuji (Biochemistry 21:6273-6279(1982)), types of amino acids are labeled with C-13 or/and N-15 such that cross peaks between (CO)-C-13(i-1) and (NH)-N-15(i) result only for pairs of sequentially adjacent amino acids of which the first is labeled with C-13 and the second with N-15. In this way, unambiguous sequence-specific assignments can be obtained for unique pairs of amino acids that occur exactly once in the sequence of the protein. To be practical, it is crucial to limit the number of differently labeled protein samples that have to be prepared while obtaining an optimal extent of labeled unique amino acid pairs. Our computer algorithm UPLABEL for optimal unique pair labeling, implemented in the program CYANA and in a standalone program, and also available through a web portal, uses combinatorial optimization to find for a given amino acid sequence labeling patterns that maximize the number of unique pair assignments with a minimal number of differently labeled protein samples. Various auxiliary conditions, including labeled amino acid availability and price, previously known partial assignments, and sequence regions of particular interest can be taken into account when determining optimal amino acid type-specific labeling patterns. The method is illustrated for the assignment of the human G-protein coupled receptor bradykinin B2 (B2R) and applied as a starting point for the backbone assignment of the membrane protein proteorhodopsin.