Development of a method allowing high resolution X-ray crystallographic analysis of proteins by site-specific heavy-atom labeling

开发一种通过位点特异性重原子标记对蛋白质进行高分辨率 X 射线晶体学分析的方法

• 批准号: 11680606
• 负责人: NAKAZAWA Takashi
• 金额: $ 2.24万
• 依托单位: Nara Women's University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1999
• 资助国家: 日本
• 起止时间: 1999 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-11680606/
• 关键词: Amio acids; heavy atom; tertiary structure of proteins; selenotryptophan; x-ray crystallography; multiwavelength anomalous dispersion (MAD); energy minimization; Monte Carlo simulated annealing; セレノトリプトファン; ペプチド合成; タンパク質; X線結晶解析; MAD法; デヒドロアミ

This research project includes synthesis of amino acids containing heavy atoms that allow high resolution X-ray crystallographic analysis of a protein labeled with a few heavy atoms at specific positions in the molecule. Although the introduction of a chalcogen atom such as selenium or tellurium into tryptophan was unsuccessful, bromine atom has been incorporated in the 5-bromotryptophan derivative (1). Because the bromine atom in 1 is much larger than hydrogen atom, there remains a need to replace the nitrogen atom in the indole ring with an atom in the same group as nitrogen if biosynthetic labeling of a protein is required : the preparation of arsenotryptophan is now in progress. An approach using semisynthesis of a protein from a small synthetic peptide containing a heavy-atom and a large peptide fragment of native origin has also been developed for the case with 1.The reliability of this method could be enhanced strongly by an algorithm allowing the calculation of the lowest-energy structure of a protein precisely. The study of tertiary-structure prediction of the S-peptide of ribonuclease A by Monte Carlo simulated annealing has proved promising because not only the experimental evidence about this peptide could be reproduced but also the electrostatic effect predicted for the side-chain groups has been supported by experiments including CD and NMR.These features evolved from this project could possibly make the structural analyses of proteins faster and more precise in resolution, although the key amino acid containing an adequate heavy atom for this purpose has not yet be prepared.

这项研究项目包括合成含有重原子的氨基酸，允许对分子中特定位置标记了几个重原子的蛋白质进行高分辨率X射线结晶学分析。虽然在色氨酸中引入硫族原子如硒或碲是不成功的，但溴原子已经被引入到5-溴色氨酸衍生物中(1)。由于1中的溴原子比氢原子大得多，如果需要对蛋白质进行生物合成标记，仍然需要用与氮属于同一基团的原子来取代吲哚环中的氮原子：目前正在进行砷色氨酸的制备。对于文献[1]中的情况，还发展了一种从含有重原子和天然大片段的小合成肽中半合成蛋白质的方法。这种方法的可靠性可以通过一种算法来大大提高，该算法允许精确地计算蛋白质的最低能量结构。用蒙特卡罗模拟退火法预测核糖核酸酶A的S肽的三级结构已被证明是很有前途的，因为不仅该肽的实验证据可以重现，而且预测的侧链的静电效应也得到了CD和NMR等实验的支持。这些特点可能使蛋白质的结构分析更快、更精确，尽管目前还没有制备出含有足够重原子的关键氨基酸。

项目成果

Y.Okamoto,M.Masuya,M.Nabeshima and T.Nakazawa: "β-Sheet formation in BPTI(16-36) by Monte Carlo simulated annealing"Chemical Physics Letters. 299巻1号. 17-24 (1999)

Y.Okamoto、M.Masuya、M.Nabeshima 和 T.Nakazawa：“通过蒙特卡罗模拟退火在 BPTI(16-36) 中形成 β-片层”《化学物理快报》第 299 卷，第 1. 17-24 期。 1999）

S.Yamazaki, M.Sakamoto, M.Doi, T.Nakazawa, Y.Kobayashi: "Synthesis of novel all-cis-functionalized cyclopropane template-assembled collagen models"Journal of Chemical Society, Perkin 1. 2001. 1870-1875 (2001)

S.Yamazaki、M.Sakamoto、M.Doi、T.Nakazawa、Y.Kobayashi：“新型全顺式功能化环丙烷模板组装胶原模型的合成”化学学会杂志，Perkin 1. 2001. 1870-1875 (

S. Yamazaki, M. Sakamoto, M. Doi, T. Nakazawa, and Y. Kobayashi: "Synthesis of novel all-cis-functionalized template-assembled collagen models"Journal Chemical Society, Perkin 1. Vol.2001. 1870-1875 (2001)

S. Yamazaki、M. Sakamoto、M. Doi、T. Nakazawa 和 Y. Kobayashi：“新型全顺式功能化模板组装胶原蛋白模型的合成”，化学学会杂志，Perkin 1. Vol.2001。

T.Nakazawa and Y.Okamoto: "Electrostatic effects on the α-helix and β-strand formation of BPTI(16-36) studied by Monte Carlo simulated annealing"Journal of Peptide Research. 54. 230-236 (1999)

T.Nakazawa 和 Y.Okamoto：“通过蒙特卡罗模拟退火研究静电对 BPTI(16-36) α-螺旋和 β-链形成的影响”《肽研究杂志》54. 230-236 (1999)。

T. Nakazawa, S. Ban, Y. Okuda, M. Masuya, A. Mitsutake, and Y. Okamoto: "A pH-dependent variation in a helix structure of the S-peptide of ribonuclease A studied by Monte Carlo simulated annealing"Biopolymers. Vol.63, No.4. 273-279 (2002)

T. Nakazawa、S. Ban、Y. Okuda、M. Masuya、A. Mitsutake 和 Y. Okamoto：“通过蒙特卡罗模拟退火研究核糖核酸酶 A S 肽螺旋结构的 pH 依赖性变化”