Regulation of G proteins by Receptor-Mimetic Peptides

点击翻译按钮获取中文摘要

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).