| Project/Area Number |
06304051
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| Research Category |
Grant-in-Aid for Co-operative Research (A)
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| Allocation Type | Single-year Grants |
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| Research Field |
Biophysics
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| Research Institution | Osaka University |
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Principal Investigator |
KATAOKA Mikio Osaka University, Faculty of Science, Associate Professor, 理学部, 助教授 (30150254)
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| Co-Investigator(Kenkyū-buntansha) |
KIHARA Hiroshi Kansai Medical University, Physics Laboratory, Professor, 医学部, 教授 (20049076)
GEKKO Kunihiko Hiroshima University, Faculty of Science, Professor, 理学部, 教授 (10023467)
KUWAJIMA Kunihiro Tokyo University, Faculty of Science, Associate Professor, 理学系研究科, 助教授 (70091444)
AKASAKA Kazuyuki Kobe University, Faculty of Science, Professor, 理学部, 教授 (50025368)
GOTO Yuji Osaka University, Faculty of Science, Associate Professor, 理学部, 助教授 (40153770)
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| Project Period (FY) |
1994 – 1995
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| Keywords | protein folding / molten globule / X-ray solution scattering / protein denturation / high pressure NMR / stopped-flow CD / chaperonine / gene engineering |
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| Research Abstract |
Solution structures of molten globules (MG) of various proteins were investigated in detail by solution X-ray scattering. The structure of MG can be classified into two categories : one is close to native structure and the other is composed of a hydrophobic core and flaring tail (s) . Some MG's are stabilized mainly by hydrophobic interaction, the other MG's are stabilized mainly by intramolecular SS bonds. The denatured states cannot be generalized by a term, random coil, because the structural diversity and variety in the denatured states were also indicated.
Kinetic studies of beta-lactoglobulin folding demonstrated the accumulation of intermediate with non-native secondary structure, which suggests the importance of non-hierarchical model for folding. Isothermal titration calorimetric measurements on MG formation indicated that the hydrophobic interaction existed in MG is almost 40% of that in native state.
It was revealed that denaturant-induced denaturation of alpha-subunit of tryp
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tophane synthase is 3 states, however its thermal denaturation is 2 states. Kinetic studies on proline mutants indicated the existence of two successive intermediates in the folding process of alpha-subunit of tryptophane synthase. Proline residues are contributed to the stability of the late intermediate.
The mechanism of target recognition by chaperonine have been investigated using MG of alpha-lactalbumin to reveal the importance of electro-static interaction.
Theoretical studies were performed on the global structures of MG and the determinant, especially the contribution of water to the MG formation. Models of MG were proposed to various proteins and the calculated scattering profiles were compared with the observed ones. Consequently the proposed theoretical method was turned out to be quite promising for the folding studies.
High pressure NMR method for protein solution was developed and applied to thermal denaturation of RNase A.It was revealed that RNase A undergoes two-state transition even under high pressure. A volume change by denaturation was negative and also a heat capacity at constant pressure was decreased significantly by addition of pressure. Adiabatic compressibility upon denaturation was measured precisely. Less
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