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Analysis of Receptor-G Protein Interactions by using Receptor Peptide Fragments

使用受体肽片段分析受体-G 蛋白相互作用

基本信息

批准号：
06680642
负责人：
WAKAMATSU Kaori
金额：
$ 1.47万
依托单位：
GUNMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06680642/
关键词：
G protein peptide mastoparan-X expression system stable isotope labeling NMR TRNOE CD 重水素化レセプターペプチドマストパラン発現

项目摘要

We obtained the following results by physicochemical analyzes of G protein-receptor interactions which ultimately aim in elucidating detailed mechanisms of signal transduction via G proteins :1.Expression of G protein alpha-subunits : We set up a system to express and easily purify recombinant Gilalpha. As high as 50 mg/L culture of Gilalpha could be prepared by Ni^<2+>-affinity chromatography of Gilalpha by attaching a histidine-tag at the N-terminus. Deuterated Gilalpha could also be obtained by this system. Aslo prepared was a K349P mutant which corresponds to the unc mutant in Gsalpha. As expected, this protein was not activated by receptor mimetics. Expression systems for Goalpha and Gsalpha were setup though the latter needs more refinements.2.Expression and stable isotope labeling of peptides : To analyze interactions between receptor-mimetic peptides and G proteins by NMR in detail, we set up a system that expresses these peptides as a fusion protein with ubiquitin and labels them with stable isotopes such as ^<13>C and ^<15>N.3.Conformation of Gilalpha-bound mastoparan-X : By TRNOE analysis in the presence of Gilalpha of mastoparan-X uniformly labeled with ^<15>N alone or both ^<13>C and ^<15>N,a precise conformation of mastoparan-X bonud to Gilalpha was obtained with rmsd as small as 0.33*. Residues from the third one to the C-terminus was found to take an alpha-helical conformation.4.Conformational change of Gilalpha upon activation by receptor mimetics : To investigate the mechanism where by stimulation by a liganded receptor enhances the GDP release from G protein, secondary structure change of Gilalpha caused by receptor mimetics was analyzed by CD.The alpha-helix content of Gilalpha was found to decrease in parallel with its activation.

通过对G蛋白-受体相互作用的物理化学分析，我们获得了以下结果，最终目的是阐明G蛋白通过信号转导的详细机制：1. G蛋白α亚基的表达：我们建立了一个表达和容易纯化重组Gilalpha的系统。通过在N-末端连接组氨酸标签的Gilalpha的Ni^<2+>-亲和层析，可以制备高达50 mg/L的Gilalpha培养物。氘代吉尔阿尔法也可以通过该系统获得。还制备了K349 P突变体，其对应于Gsalpha中的unc突变体。正如预期的那样，这种蛋白质不被受体模拟物激活。2.多肽的表达和稳定同位素标记：为了通过NMR详细分析受体模拟肽与G蛋白之间的相互作用，我们建立了将这些肽表达为与泛素的融合蛋白并用稳定同位素如^ C和^ N标记它们的系统<13>。<15>3.结合Gilalpha的mastoparan-X的构象：通过TRNOE分析，在Gilalpha存在下，用^<15>N单独或^<13>C和^<15>N均匀标记mastoparan-X，得到mastoparan-X结合Gilalpha的精确构象，rmsd小至0.33*。发现从第三个残基到C-末端的残基采取α-螺旋构象。4.受体模拟物激活后Gilalpha的构象变化：为了研究通过配体受体的刺激增强G蛋白的GDP释放的机制，通过CD分析受体模拟物引起的Gilalpha二级结构变化。发现Gilalpha的螺旋含量与其活化平行地降低。