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Regulation of G proteins by Receptor-Mimetic Peptides

受体模拟肽对 G 蛋白的调节

基本信息

批准号：
03044029
负责人：
WAKAMATSU Kaori
金额：
$ 6.4万
依托单位：
Gunma University
依托单位国家：
日本
项目类别：
Grant-in-Aid for international Scientific Research
财政年份：
1991
资助国家：
日本
起止时间：
1991 至 1992
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-03044029/
关键词：
G Protein Adrenergic Receptor Muscarinic Receptor CD NMR Conformation Phospholipid Bilayer Peptide Lipid Interaction ムスカリンレセプタ-コンフォメ-ション

项目摘要

1. Preparation and Activity of Receptor-Mimetic Peptides : We synthesized peptides corresponding to intracellular loops of b-adrenergic receptors muscarinic riceptors. beta-receptor peptides activated Gs protein selectively when it is alkylated at its N-terminus.2. Preparation of Non-labeled and Stable Isotope-Labelled G proteins : We succeeded in large-scale preparation of Gsalpha and Gilalpha protein (> 10 mg/liter). We also obtained Gilalpha proteins that are either uniformly-labelled with ^<15>N or selectively-labelled with [^<15>15n]Trp.3. Preparation of Perdeuterated Phospholipids : We synthesized perdeuterated phosphatidylcholine (DPPC-d_<80>) and perdeuterated phosphatidylserine (DLPS-d_<54>).4. Conformation of Peptides Bound to Phospholipid Membrane and G Protein : We determined conformation of receptor-mimetic peptides bound to phospholipid bilayer and to G proteins by CD, transferred NOE (NMR) combine with distance-geometry and simulated annealing calculations. Both a turkey beta-receptor peptide and mastoparan-X took amphiphilic alpha-helical conformations when bound to phospholipid membrane and to Gs protein.5. Structural Change of G Protein Upon Activation By NMR, we analyzed conformational change of Gilalpha protein activation. More than 30 % of ^1H-^<15>N correlation peaks of the uniformly 15N labeled protein were observed in HSQC spectra irrespective of the high molecular weight of the protein (41 kd). Upon the activation by Al^<3+>, Mg^<2+> and F^-, many resonances shifted, which indicates that the conformation change associated with the activation is not localized.6. Simple and Direct Measurement of Phospholipid Hydration in Liquid Water : During our study to monitor the binding of peptide to phospholipid membrane, we found that the hydration of phospholipid can be easily and directly detected by quartz-crystal microbalance. We then systematically the hydration under various conditions (phase of water, temperature, ionic strength).

1.受体模拟肽的制备和活性：我们合成了对应于β-肾上腺素能受体毒蕈碱受体胞内环的肽。当Gs蛋白的N端烷基化时，β受体肽选择性地激活Gs蛋白。2．非标记和稳定同位素标记的 G 蛋白的制备：我们成功大规模制备了 Gsalpha 和 Gilalpha 蛋白（> 10 毫克/升）。我们还获得了用^ 15 N均匀标记或用[^ 15 15n]Trp.3选择性标记的Gilalpha蛋白。全氘化磷脂的制备：我们合成了全氘化磷脂酰胆碱(DPPC-d_<80>)和全氘化磷脂酰丝氨酸(DLPS-d_<54>)。 4．与磷脂膜和 G 蛋白结合的肽的构象：我们通过 CD、转移 NOE (NMR) 结合距离几何和模拟退火计算确定了与磷脂双层和 G 蛋白结合的受体模拟肽的构象。火鸡β-受体肽和mastoparan-X在与磷脂膜和Gs蛋白结合时均呈现两亲性α-螺旋构象。5． G 蛋白激活后的结构变化通过 NMR，我们分析了 Gilalpha 蛋白激活后的构象变化。无论蛋白质的高分子量(41kd)如何，在HSQC谱中观察到均匀15N标记蛋白质的超过30％的^ 1 H-^ 15 N相关峰。 Al^<3+>、Mg^<2+>和F^-激活后，许多共振发生转移，这表明与激活相关的构象变化不是局部的。6．简单直接地测量液态水中的磷脂水合：在监测肽与磷脂膜的结合的研究中，我们发现石英晶体微天平可以轻松直接地检测磷脂的水合。然后我们系统地在各种条件（水相、温度、离子强度）下进行水合作用。