通过组合鸟枪法扫描诱变评估蛋白质结合位点的分子内协同性。

Intramolecular cooperativity in a protein binding site assessed by combinatorial shotgun scanning mutagenesis.

作者信息

Pál Gábor, Ultsch Mark H, Clark Kevin P, Currell Bridget, Kossiakoff Anthony A, Sidhu Sachdev S

机构信息

Department of Biochemistry and Molecular Biology and Institute for Biophysical Dynamics, Cummings Life Sciences Center, University of Chicago, 920 East 58th Street, Chicago, IL 60637, USA.

出版信息

J Mol Biol. 2005 Apr 1;347(3):489-94. doi: 10.1016/j.jmb.2005.01.040.

DOI:10.1016/j.jmb.2005.01.040

PMID:15755445

Abstract

Combinatorial shotgun alanine-scanning was used to assess intramolecular cooperativity in the high affinity site (site 1) of human growth hormone (hGH) for binding to its receptor. A total of 19 side-chains were analyzed and statistically significant data were obtained for 145 of the 171 side-chain pairs. The analysis revealed that 90% of the side-chain pairs exhibited no statistically significant pair interactions, and the remaining 10% of side-chain pairs exhibited only small interactions corresponding to cooperative interaction energies with magnitudes less than 0.4 kcal/mol. The statistical predictions were tested by measuring affinities for purified mutant proteins and were found to be accurate for five of six side-chain pairs tested. The results reveal that hGH site 1 behaves in a highly additive manner and suggest that shotgun scanning should be useful for assessing cooperative effects in other protein-protein interactions.

摘要

采用组合鸟枪法丙氨酸扫描来评估人生长激素（hGH）与受体结合的高亲和力位点（位点1）内的分子内协同性。共分析了19个侧链，在171个侧链对中的145个获得了具有统计学意义的数据。分析表明，90%的侧链对没有表现出具有统计学意义的配对相互作用，其余10%的侧链对仅表现出较小的相互作用，对应于协同相互作用能量，其大小小于0.4千卡/摩尔。通过测量纯化突变蛋白的亲和力对统计预测进行了测试，发现所测试的六个侧链对中有五个预测准确。结果表明，hGH位点1以高度加和的方式起作用，并表明鸟枪法扫描对于评估其他蛋白质-蛋白质相互作用中的协同效应应该是有用的。