1992 Fiscal Year Final Research Report Summary
Synthesis of Ca^<2+> -responsive DNA-binding Allosteric Protein
| Project/Area Number |
03453117
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
高分子合成
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| Research Institution | KYOTO UNIVERSITY |
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Principal Investigator |
IMANISHI Yukio Kyoto Univ.・Faculty of Engineering, Professor, 工学部, 教授 (00025991)
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| Co-Investigator(Kenkyū-buntansha) |
KIMURA Shunsaku Kyoto Univ.・Faculty of Engineering, Lecturer, 工学部, 講師 (80150324)
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| Project Period (FY) |
1991 – 1992
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| Keywords | Protein model / Super-secondary structure / alpha helix / Cyclic peptide / Peptide engineering / amphiphilic peptide / DNA-binding protein |
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| 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+>.
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