UNNATURAL AMINO ACID INCORPORATION INTO PROTEINS AND QUANTIFICATION THEROF

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 最近，彼得·舒尔茨教授的实验室(斯克里普斯)率先提出了一种方法，允许在蛋白质序列中的任何位置特定地结合非天然氨基酸。这项技术已经被证明是有用的，用荧光标记蛋白质，13C和15N，以及化学交联非天然氨基酸，用于体内和体外结构功能研究。通过使用正交的jannaschii tRNA/tRNA-合成酶对，即tRNA和合成酶都不与内源E.colitRNA或氨基酰基tRNA合成酶交叉反应，可以使用仅由正交tRNA识别的密码子在蛋白质的任何位置引入非自然的氨基酸残基。通过与斯克里普斯的舒尔茨实验室的合作，保罗·奥尔蒂斯·德·蒙特拉诺实验室已经能够在内部获得这种标记技术，并在最近成功地使用它标记了几种含有血红素的蛋白质，为进一步的生物物理研究做准备。不幸的是，缺乏关于非天然氨基酸的掺入程度和相对表达水平的信息，限制了这项技术的应用。为此，我们希望利用加州大学旧金山分校药物化学系的质谱学设施来获得非天然氨基酸在各种蛋白质中的结合程度和位置，并确定它们的相对表达水平。这一知识不仅将推动我们的工作，还将使这项强大的技术得到更广泛的采用。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Recently, an approach pioneered in Prof. Peter Schultz lab (Scripps) allows for the site-specific incorporation of an unnatural amino acid anywhere within a protein sequence. This technology has already proven useful for labeling proteins with fluorescent, 13C and 15N, and chemical crosslinking unnatural amino acids for in vivo and in vitro structure-function studies. By using an orthogonal M. jannaschii tRNA/tRNA-synthetase pair, that is one where neither the tRNA nor the synthetase cross-reacts with the endogenous E.coli tRNA's or amino acyl tRNA sythetases, it is possible to introduce an unnatural amino acid residue at any position in the protein using a codon that is only recognized by the orthogonal tRNA. Through collaboration with the Schultz lab at Scripps, the Paul Ortiz de Montellano lab has been able to acquire this labeling technology in house and has recently used it to successfully label several heme containing proteins in preparation for further biophysical studies. Unfortunately, lack of information as to the extent of incorporation of the unnatural amino acid and relative expression levels has limited the application of this technology. To that end, we hope to utilize the UCSF Department of Pharmaceutical Chemistry Mass Spectrometry Facility to access the extent and location of incorporation of the unnatural amino acids into various proteins, and also determine their relative expression levels. This knowledge will not only further our work, but will also allow for more widespread adoption of this powerful technology.