1992 Fiscal Year Final Research Report Summary
Regulation of G proteins by Receptor-Mimetic Peptides
| Project/Area Number |
03044029
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| Research Category |
Grant-in-Aid for international Scientific Research
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| Allocation Type | Single-year Grants |
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| Section | Joint Research |
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| Research Institution | Gunma University |
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Principal Investigator |
WAKAMATSU Kaori Faculty of Engineering, Gunma University, 工学部, 助教授 (40222426)
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| Co-Investigator(Kenkyū-buntansha) |
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
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| Project Period (FY) |
1991 – 1992
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| Keywords | G Protein / Adrenergic Receptor / Muscarinic Receptor / CD / NMR / Conformation / Phospholipid Bilayer / Peptide / Lipid Interaction |
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| Research Abstract |
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).
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