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。 由Peter Schultz教授(斯克里普斯)首创的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。这项技术已被证明是有用的，用荧光标记蛋白质，13C和15N，以及化学交联的非天然氨基酸，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合成酶交叉反应的对，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。Ortiz de Monellano实验室从舒尔茨实验室获得了这项标记技术，并正在使用它标记用于生物物理和生物化学研究的各种含有血红素的蛋白质。有关非天然氨基酸的结合程度和蛋白质的相对表达水平的信息可能会严重限制这项技术的应用。为此，我们利用加州大学旧金山分校的质谱学设备来确定非天然氨基酸在蛋白质中的结合程度和位置，并确定它们的相对表达水平。这一知识不仅对我们的工作至关重要，而且将使更广泛地采用这项强大的技术成为可能。

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. An approach pioneered by Prof. Peter Schultz (Scripps) allows for the site-specific incorporation of an unnatural amino acid anywhere within a protein sequence. This technology has 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, i.e., one where neither the tRNA nor the synthetase cross-reacts with the endogenous E.coli tRNA's or amino acyl tRNA synthetases, 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. The Ortiz de Montellano lab acquired this labeling technology courtesy of the Schultz lab and is using it to label diverse heme containing proteins for biophysical and biochemical studies. Information on the extent of incorporation of the unnatural amino acid and the relative expression level of the protein can severely limit the application of this technology. For that reason, we utilize the UCSF Mass Spectrometry Facility to determine the extent and site of incorporation of the unnatural amino acids into the proteins, and to determine their relative expression levels. This knowledge is not only critical for our work, but will make possible the more widespread adoption of this powerful technology.