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

该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有几个重原子的蛋白质进行高分辨率X射线晶体学分析。虽然硫族元素原子如硒或碲引入色氨酸是不成功的，但溴原子已被引入5-溴色氨酸衍生物（1）。由于1中的溴原子比氢原子大得多，如果需要生物合成标记蛋白质，仍然需要用与氮相同基团的原子取代吲哚环中的氮原子：砷色氨酸的制备现在正在进行中。本文还提出了一种由含重原子的合成肽和天然肽片段半合成蛋白质的方法，该方法可以通过精确计算蛋白质的最低能量结构的算法来增强其可靠性。用Monte Carlo模拟退火法预测核糖核酸酶A的S肽的三级结构，不仅可以重现有关该肽的实验证据，而且可以预测其侧链的静电效应，链基团已经得到了包括CD和NMR在内的实验的支持。从这个项目中发展出来的这些特征可能会使蛋白质的结构分析更快，更精确，虽然含有足够重原子的关键氨基酸尚未制备。

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.