A unified genetic, computational and experimental framework identifies functionally relevant residues of the homing endonuclease I-BmoI.
A unified genetic, computational and experimental framework identifies functionally relevant residues of the homing endonuclease I-BmoI.
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DOI：
10.1093/nar/gkp1223
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发表时间：
2010-04
影响因子：
14.9
通讯作者：
Edgell DR
中科院分区：
文献类型：
--
作者：
Kleinstiver BP;Fernandes AD;Gloor GB;Edgell DR
深入了解蛋白质的结构和功能最好是通过实验，结构和生物信息学数据的综合获得。在这里，我们概述了一个框架，我们称之为MUSE（互信息，单基因进化和结构指导的阐明），这有利于识别以前未知的残基，是相关的功能的GIY-YIG归巢核酸内切酶I-BmoI。我们的方法综合了三种类型的数据：互信息分析，确定共同进化的残基内的GIY-YIG催化结构域;一个单基因的进化策略，确定超和亚突变的I-BmoI残基;和解释的单基因和共同进化的数据，使用同源模型。特别是，我们确定新的位置内的GIY-YIG域功能上的重要性。原理验证实验表明，非保守的I71在功能上是相关的，I71 N突变体积累了一个有切口的切割中间体。此外，I-BmoI的催化、接头和C-末端结构域内的许多其他位置被认为对功能很重要。我们的研究结果代表了一个平台，在此基础上进行I-BmoI和其他含GIY-YIG的蛋白质的未来研究，并证明MUSE可以成功地识别新的功能关键残基，这些残基在广泛研究的90个氨基酸的小结构域内的传统结构-功能分析中会被忽略。
Insight into protein structure and function is best obtained through a synthesis of experimental, structural and bioinformatic data. Here, we outline a framework that we call MUSE (mutual information, unigenic evolution and structure-guided elucidation), which facilitated the identification of previously unknown residues that are relevant for function of the GIY-YIG homing endonuclease I-BmoI. Our approach synthesizes three types of data: mutual information analyses that identify co-evolving residues within the GIY-YIG catalytic domain; a unigenic evolution strategy that identifies hyper- and hypo-mutable residues of I-BmoI; and interpretation of the unigenic and co-evolution data using a homology model. In particular, we identify novel positions within the GIY-YIG domain as functionally important. Proof-of-principle experiments implicate the non-conserved I71 as functionally relevant, with an I71N mutant accumulating a nicked cleavage intermediate. Moreover, many additional positions within the catalytic, linker and C-terminal domains of I-BmoI were implicated as important for function. Our results represent a platform on which to pursue future studies of I-BmoI and other GIY-YIG-containing proteins, and demonstrate that MUSE can successfully identify novel functionally critical residues that would be ignored in a traditional structure-function analysis within an extensively studied small domain of ∼90 amino acids.
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影响因子：
5.8
作者：
Dunn, S. D.;Wahl, L. M.;Gloor, G. B.
通讯作者：
Gloor, G. B.
影响因子：
64.8
作者：
Ashworth, Justin;Havranek, James J.;Baker, David
通讯作者：
Baker, David
影响因子：
14.9
作者：
Landau M;Mayrose I;Rosenberg Y;Glaser F;Martz E;Pupko T;Ben-Tal N
通讯作者：
Ben-Tal N
影响因子：
3
作者：
Behrsin, CD;Brandl, CJ;Wahl, LM
通讯作者：
Wahl, LM
影响因子：
5.8
作者：
Arnold, K;Bordoli, L;Schwede, T
通讯作者：
Schwede, T