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Synthesis of Ca^<2+> -responsive DNA-binding Allosteric Protein

Research Project

Project/Area Number 03453117
Research Category

Grant-in-Aid for General Scientific Research (B)

Allocation TypeSingle-year Grants
Research Field 高分子合成
Research InstitutionKYOTO UNIVERSITY

Principal Investigator

IMANISHI Yukio  Kyoto Univ.・Faculty of Engineering, Professor, 工学部, 教授 (00025991)

Co-Investigator(Kenkyū-buntansha) KIMURA Shunsaku  Kyoto Univ.・Faculty of Engineering, Lecturer, 工学部, 講師 (80150324)
Project Period (FY) 1991 – 1992
Project Status Completed (Fiscal Year 1992)
Budget Amount *help
¥6,400,000 (Direct Cost: ¥6,400,000)
Fiscal Year 1992: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1991: ¥5,000,000 (Direct Cost: ¥5,000,000)
KeywordsProtein model / Super-secondary structure / alpha helix / Cyclic peptide / Peptide engineering / amphiphilic peptide / DNA-binding protein / アロステリック効果 / ヘリックスバンドル / 両親媒性構造 / カルシウムイオン / 錯体形成
Research Abstract

DNA-binding proteins are classified into a few structural motifs. One of them is an assembly of two chains with many basic residues, which are brought close with each other by the extended peptide fragment called "Leu zipper". In the present study, we have used cyclic peptides which were expected to mimic function of "Leu zipper". Two chains of dodecapeptides were connected to cyclic octapeptides, cyclo(Leu-Sar-Lys(Z)-Sar)_2 (C8KS) and cyclo(Leu-Sar-Lys(CHO)-Sar-Leu-Sar-Glu(OBzl)-Sar) (C8KE). Both cyclic peptides formed a complex with Ca^<2+>. The protecting groups of the cyclic peptides were removed, and two chains of Boc-Glu-Napala-Leu-Aib-(Lys-Aib-Leu-Aib)_2 - (Napala represents 2-naphthylalanine) were bound to C8KS (F12-C8KS), and Ac-Glu(OMe)-Trp-Leu-Aib-(Lys-Aib-Leu-Aib)_2-and-(Leu-Aib-Glu(ONe)-Aib)_2-Lew-Aib-Ant (Ant represents an anthryl derivative) to C8KE (CH2). CD and fluorescence spectroscopy revealed that CH2 took a super-secondary structure of association of two helical chains in a buffer solution. The structure should be stable due to the following two reasons: i) two chains protrude over the same side of the cyclic skeleton, and ii) the dodecapeptides take an amphiphilic alpha helical conformation.
With the addition of Lambda DNA to CH2 in alpha buffer solution, the fluorescence from the Trp residue was quenched, and a new signal from the anthryl group appeared at a longer wavelength. Thus, CH2 interacted with Lambda DNA, probably intercalating the indolyl and anthryl groups into base pairs of the DNA. On the other hand, F12-C8KS and a cyclic peptide having one chain did not interact with DNA. Therefore, the super-secondary structure of CH2 should be critical for the interaction with DNA. Interestingly, the interaction mode of CH2 and DNA was changed by the presence of Ca^<2+>.

Report

(3 results)
  • 1992 Annual Research Report   Final Research Report Summary
  • 1991 Annual Research Report
  • Research Products

    (1 results)

All Other

All Publications (1 results)

  • [Publications] Yukio Imanishi and Shunsaku Kimura(分担執筆): "Fundamental Investigations on the Creation of Biofunctional" 化学同人, 9 (1991)

    • Related Report
      1991 Annual Research Report

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Published: 1991-04-01   Modified: 2016-04-21  

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