Using peptide arrays to define nuclear carrier binding sites on nucleoporins
Using peptide arrays to define nuclear carrier binding sites on nucleoporins
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DOI：
10.1016/j.ymeth.2006.06.011
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发表时间：
2006-08-01
期刊：
影响因子：
4.8
通讯作者：
Moore, Mary Shannon
中科院分区：
文献类型：
--
作者：
Cushman, Ian;Palzkill, Timothy;Moore, Mary Shannon
在肽SPOT阵列技术中，不同的肽阵列在纤维素膜上合成，并以共价连接到纤维素膜上。在该技术的一种用法中，这些肽在覆盖试验中筛选，以确定哪些短序列包含相互作用蛋白质的结合位点。通过制备覆盖蛋白质整个序列的重叠肽，可以识别第二种蛋白质的蛋白质上的所有结合域。我们利用肽SPOT阵列技术鉴定了结合核载体importin- β的核孔复合物蛋白（也称为核孔蛋白或Nups）内的短氨基酸序列。其他人的结晶研究表明，核载体如importin- β与苯丙氨酸-甘氨酸（FG）重复序列结合，在核孔蛋白家族的序列中存在许多拷贝。与此一致的是，我们发现通过该技术鉴定的大多数（但不是全部）importin-beta的Nup结合位点包含Fx、FG、FxFG、FxFx或GLFG序列，尽管并非所有这些序列都与importin-beta结合。肽点阵列替代研究证实了苯丙氨酸在FG重复序列中的关键作用，并在一些重复序列的侧翼发现了赖氨酸残基，这对于importin- β与这些重复序列的结合至关重要。除了这些预期的importin-beta结合序列外，我们还发现了多个缺乏规范的FG重复序列的肽，这些重复序列强烈结合importin-beta，这表明额外的Nup序列可能形成importin-beta的结合位点。(c) 2006爱思唯尔公司版权所有。
In the peptide SPOT array technique, an array of different peptides are synthesized on, and covalently linked to, cellulose membranes. In one usage of this technique, these peptides are screened in an overlay assay to determine which short sequence(s) contains a binding site for an interacting protein. By preparing overlapping peptides that cover the entire sequence of a protein, all of the binding domains on the protein for a second protein can be identified. We have utilized the peptide SPOT array technique to identify the short amino acid sequences within nuclear pore complex proteins (also known as nucleoporins or Nups) that bind the nuclear carrier importin-beta. Crystallization studies by others have indicated that nuclear carriers such as importin-beta bind to phenylalanine-glycine (FG) repeats present in numerous copies in the sequences of a family of nucleoporins. Consistent with this, we found that most (but not all) of the Nup binding sites for importin-beta identified by this technique contain Fx, FG, FxFG, FxFx, or GLFG sequences, although not all such sequences bound importin-beta. Peptide SPOT array substitution studies confirmed a crucial role for the phenylalanine in FG repeats and identified a lysine residue flanking some repeats that is crucial for importin-beta binding to those repeats. In addition to these expected binding sequences for importin-beta, we found multiple instances of a peptide lacking a canonical FG repeat that strongly bound importin-beta, indicating that additional Nup sequences may form binding sites for importin-beta. (c) 2006 Elsevier Inc. All rights reserved.