https://scholars.lib.ntu.edu.tw/handle/123456789/414371
標題: | Combining secondary-structure and protein solvent-accessibility predictions in methionine substitution for anomalous dispersion | 作者: | Wu H.-Y. Cheng Y.-S. |
關鍵字: | anomalous dispersion;Arabidopsis thaliana thylakoid lumen protein;MAD;methionine substitution;multi-wavelength anomalous diffraction;protein solvent-accessible surface-area prediction;SAD;secondary-structure prediction;single-wavelength anomalous diffraction;solvent accessibility | 公開日期: | 2014 | 卷: | 70 | 期: | 3 | 起(迄)頁: | 378-383 | 來源出版物: | Acta Crystallographica Section F:Structural Biology Communications | 摘要: | In X-ray crystallographic analysis, the single-wavelength and multi-wavelength anomalous diffraction (SAD and MAD) methods have been widely used in order to solve the phase problem. Selenium-labelled methionine has been shown to be very effective for anomalous dispersion phasing, and at least one selenomethionine is required for every 100 amino acids. Some proteins, such as the Arabidopsis thaliana thylakoid lumen protein AtTLP18.3, can be overexpressed in an Escherichia coli system and high-quality protein crystals can be obtained. However, AtTLP18.3 contains no methionine residues, and site-directed mutagenesis was required in order to introduce methionine residues into the protein. A criterion for the mutated residues is that they should avoid affecting the structure and function. In this study, several leucine and isoleucine residues were selected for methionine substitution by combining secondary-structure and solvent-accessibility predictions. From the secondary-structure prediction, mutated residues were first determined in the coil or loop regions at the junction of two secondary structures. Since leucine and isoleucine residues are hydrophobic and are normally buried within the protein core, these residues should have a higher solvent-accessibility prediction so that they would be partially buried or exposed in the protein. In addition, five residues (Leu107, Leu202, Ile133, Leu128 and Ile159) of AtTLP18.3 were mutated to methionine residues. After overexpression and purification, only two single-mutant lines, L128M and I159M, could be crystallized. Finally, a double-mutation line of truncated AtTLP18.3 with L128M and I159M mutations was constructed. The structure of the double mutant AtTLP18.3 protein was resolved using the single-wavelength anomalous diffraction method at 2.6 ? resolution. The results indicated that a combination of secondary-structure and solvent-accessibility prediction for methionine substitution is a useful method in SAD and MAD phasing. ? 2014 International Union of Crystallography. |
URI: | https://scholars.lib.ntu.edu.tw/handle/123456789/414371 | ISSN: | 2053230X | DOI: | 10.1107/S2053230X14001897 |
顯示於: | 植物科學研究所 |
在 IR 系統中的文件,除了特別指名其著作權條款之外,均受到著作權保護,並且保留所有的權利。