Affinity maturation of a portable Fab-RNA module for chaperone-assisted RNA crystallography.
Affinity maturation of a portable Fab-RNA module for chaperone-assisted RNA crystallography.
复制标题
DOI：
10.1093/nar/gkx1292
复制
发表时间：
2018-03-16
影响因子：
14.9
通讯作者：
Piccirilli JA
中科院分区：
文献类型：
--
作者：
Koirala D;Shelke SA;Dupont M;Ruiz S;DasGupta S;Bailey LJ;Benner SA;Piccirilli JA
Antibody fragments such as Fabs possess properties that can enhance protein and RNA crystallization and therefore can facilitate macromolecular structure determination. In particular, Fab BL3–6 binds to an AAACA RNA pentaloop closed by a GC pair with ∼100 nM affinity. The Fab and hairpin have served as a portable module for RNA crystallization. The potential for general application make it desirable to adjust the properties of this crystallization module in a manner that facilitates its use for RNA structure determination, such as ease of purification, surface entropy or binding affinity. In this work, we used both in vitro RNA selection and phage display selection to alter the epitope and paratope sides of the binding interface, respectively, for improved binding affinity. We identified a 5′-GNGACCC-3′ consensus motif in the RNA and S97N mutation in complimentarity determining region L3 of the Fab that independently impart about an order of magnitude improvement in affinity, resulting from new hydrogen bonding interactions. Using a model RNA, these modifications facilitated crystallization under a wider range of conditions and improved diffraction. The improved features of the Fab–RNA module may facilitate its use as an affinity tag for RNA purification and imaging and as a chaperone for RNA crystallography.
登录
查看更多内容
影响因子：
14.9
作者：
Goujon M;McWilliam H;Li W;Valentin F;Squizzato S;Paern J;Lopez R
通讯作者：
Lopez R
影响因子：
5.7
作者：
Hamma, T;Ferré-D'Amaré, AR
通讯作者：
Ferré-D'Amaré, AR
模因套件：用于发现和搜索的工具。
影响因子：
14.9
作者：
Bailey TL;Boden M;Buske FA;Frith M;Grant CE;Clementi L;Ren J;Li WW;Noble WS
通讯作者：
Noble WS
含有 G-四链体的 RNA 会激活类 GFP 荧光团中的荧光。
影响因子：
14.8
作者：
Huang, Hao;Suslov, Nikolai B.;Li, Nan-Sheng;Shelke, Sandip A.;Evans, Molly E.;Koldobskaya, Yelena;Rice, Phoebe A.;Piccirilli, Joseph A.
通讯作者：
Piccirilli, Joseph A.
影响因子：
5.6
作者：
Ferré-D'Amaré, AR;Doudna, JA
通讯作者：
Doudna, JA