Regulation of G proteins by Receptor-Mimetic Peptides
Grant-in-Aid for international Scientific Research
|Allocation Type||Single-year Grants|
|Research Institution||Gunma University|
WAKAMATSU Kaori Faculty of Engineering, Gunma University, 工学部, 助教授 (40222426)
OYA Masanao Faculty of Engineering, Gunma University, 教授 (50008489)
TSUTOMU HIGA Department of Pharmacology, University of, Associate
HIGASHIJIMA Tsutomu University of Texas Southwestern Medical Center at Dallas
|Project Period (FY)
1991 – 1992
Completed(Fiscal Year 1992)
|Budget Amount *help
¥10,000,000 (Direct Cost : ¥10,000,000)
Fiscal Year 1992 : ¥5,000,000 (Direct Cost : ¥5,000,000)
Fiscal Year 1991 : ¥5,000,000 (Direct Cost : ¥5,000,000)
|Keywords||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|
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).
Research Output (15results)